Methods and compositions for the specific inhibition of glycolate oxidase (hao1) by double-stranded rna

ABSTRACT

This invention relates to compounds, compositions, and methods useful for reducing Glycolate Oxidase (HAO1) target RNA and protein levels via use of dsRNAs, e.g., Dicer substrate siRNA (DsiRNA) agents.

RELATED APPLICATIONS

The present application claims priority to, and the benefit under 35U.S.C. §119(e) of U.S. provisional patent application No. 61/921,181,entitled “Methods and Compositions for the Specific Inhibition ofGlycolate Oxidase (HAO1) by Double-Stranded RNA,” filed Dec. 27, 2013,and of U.S. provisional application No. 61/937,838, entitled “Methodsand Compositions for the Specific Inhibition of Glycolate Oxidase (HAO1)by Double-Stranded RNA,” filed Feb. 10, 2014. The entire contents of theaforementioned patent applications are incorporated herein by thisreference.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of Glycolate Oxidase (HAO1)gene expression and/or activity.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled “301937_94015WO_seq_listing”, was created on Dec. 23, 2014 and is 2.87 MB in size.The information in the electronic format of the Sequence Listing is partof the present application and is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Primary hyperoxaluria Type 1 (“PH1”) is a rare autosomal recessiveinborn error of glyoxylate metabolism, caused by a deficiency of theliver-specific enzyme alanine:glyoxylate aminotransferase. The disorderresults in overproduction and excessive urinary excretion of oxalate,causing recurrent urolithiasis and nephrocalcinosis. As glomerularfiltration rate declines due to progressive renal involvement, oxalateaccumulates leading to systemic oxalosis. The diagnosis is based onclinical and sonographic findings, urine oxalate assessment, enzymologyand/or DNA analysis. While early conservative treatment has aimed tomaintain renal function, in chronic kidney disease Stages 4 and 5, thebest outcomes to date have been achieved with combined liver-kidneytransplantation (Cochat et al. Nephrol Dial Transplant 27: 1729-36).

PH1 is the most common form of primary hyperoxaluria and has anestimated prevalence of 1 to 3 cases per 1 million population and anincidence rate of approximately 1 case per 120,000 live births per yearin Europe (Cochat et al. Nephrol Dial Transplant 10 (Suppl 8): 3-7; vanWoerden et al. Nephrol Dial Transplant 18: 273-9). It accounts for 1 to2% of cases of pediatric end-stage renal disease (ESRD), according toregistries from Europe, the United States, and Japan (Harambat et al.Clin J Am Soc Nephrol 7: 458-65), but it appears to be more prevalent incountries in which consanguineous marriages are common (with aprevalence of 10% or higher in some North African and Middle Easternnations; Kamoun and Lakhoua Pediatr Nephrol 10: 479-82; see Cochat andRumsby N Engl J Med 369(7):649-58).

Glycolate oxidase (the product of the HAO1, for “hydroxyacid oxidase 1”,gene) is the enzyme responsible for converting glycolate to glyoxylatein the mitochondrial/peroxisomal glycine metabolism pathway in the liverand pancreas. While glycolate is a harmless intermediate of the glycinemetabolism pathway, accumulation of glyoxylate (via, e.g., AGT1mutation) drives oxalate accumulation, which ultimately results in thePH1 disease.

Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35nucleotides have been described as effective inhibitors of target geneexpression in mammalian cells (Rossi et al., U.S. Pat. No. 8,084,599 andU.S. Patent Application No. 2005/0277610). dsRNA agents of such lengthare believed to be processed by the Dicer enzyme of the RNA interference(RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA”(“DsiRNA”) agents. Additional modified structures of DsiRNA agents werepreviously described (Rossi et al., U.S. Patent Application No.2007/0265220). Effective extended forms of Dicer substrates have alsorecently been described (Brown, U.S. Pat. No. 8,349,809 and U.S. Pat.No. 8,513,207).

BRIEF SUMMARY OF THE INVENTION

The present invention is based, at least in part, upon theidentification of HAO1 as an attractive target for dsRNA-based knockdowntherapies. In particular, provided herein are nucleic acid agents thattarget and reduce expression of HAO1. Such compositions contain nucleicacids such as double stranded RNA (“dsRNA”), and methods for preparingthem. The nucleic acids of the invention are capable of reducing theexpression of a target Glycolate Oxidase gene in a cell, either in vitroor in a mammalian subject.

In one aspect, the invention provides a nucleic acid possessing anoligonucleotide strand of 15-80 nucleotides in length, where theoligonucleotide strand is sufficiently complementary to a target HAO1mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides ofthe oligonucleotide strand length to reduce HAO1 target mRNA expressionwhen the nucleic acid is introduced into a mammalian cell.

Another aspect of the invention provides a nucleic acid possessing anoligonucleotide strand of 19-80 nucleotides in length, where theoligonucleotide strand is sufficiently complementary to a target HAO1mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides ofthe oligonucleotide strand length to reduce HAO1 target mRNA expressionwhen the nucleic acid is introduced into a mammalian cell.

In one embodiment, the oligonucleotide strand is 19-35 nucleotides inlength.

An additional aspect of the invention provides a double stranded nucleicacid (dsNA) possessing first and second nucleic acid strands includingRNA, where the first strand is 15-66 nucleotides in length and thesecond strand of the dsNA is 19-66 nucleotides in length, where thesecond oligonucleotide strand is sufficiently complementary to a targetHAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15nucleotides of the second oligonucleotide strand length to reduce HAO1target mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell.

Another aspect of the invention provides a double stranded nucleic acid(dsNA) possessing first and second nucleic acid strands, where the firststrand is 15-66 nucleotides in length and the second strand of the dsNAis 19-66 nucleotides in length, where the second oligonucleotide strandis sufficiently complementary to a target HAO1 mRNA sequence of SEQ IDNOs: 1921-2304 along at least 19 nucleotides of the secondoligonucleotide strand length to reduce HAO1 target mRNA expression whenthe double stranded nucleic acid is introduced into a mammalian cell.

A further aspect of the invention provides a double stranded nucleicacid (dsNA) possessing first and second nucleic acid strands, where thefirst strand is 15-66 nucleotides in length and the second strand of thedsNA is 19-66 nucleotides in length, where the second oligonucleotidestrand is sufficiently complementary to a target HAO1 mRNA sequence ofSEQ ID NOs: 1921-2304 along at least 19 nucleotides of the secondoligonucleotide strand length to reduce HAO1 target mRNA expression, andwhere, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs:1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a sitebetween positions 9 and 10 of the sequence, when the double strandednucleic acid is introduced into a mammalian cell.

Another aspect of the invention provides a dsNA molecule, consisting of:(a) a sense region and an antisense region, where the sense region andthe antisense region together form a duplex region consisting of 25-66base pairs and the antisense region includes a sequence that is thecomplement of a sequence of SEQ ID NOs: 1921-2304; and (b) from zero totwo 3′ overhang regions, where each overhang region is six or fewernucleotides in length, and where, starting from the 5′ end of the HAO1mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2cleaves the mRNA at a site between positions 9 and 10 of the sequence,when the double stranded nucleic acid is introduced into a mammaliancell.

An additional aspect of the invention provides a double stranded nucleicacid (dsNA) possessing first and second nucleic acid strands and aduplex region of at least 25 base pairs, where the first strand is 25-65nucleotides in length and the second strand of the dsNA is 26-66nucleotides in length and includes 1-5 single-stranded nucleotides atits 3′ terminus, where the second oligonucleotide strand is sufficientlycomplementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304along at least 19 nucleotides of the second oligonucleotide strandlength to reduce HAO1 target gene expression when the double strandednucleic acid is introduced into a mammalian cell.

Another aspect of the invention provides a double stranded nucleic acid(dsNA) possessing first and second nucleic acid strands and a duplexregion of at least 25 base pairs, where the first strand is 25-65nucleotides in length and the second strand of the dsNA is 26-66nucleotides in length and includes 1-5 single-stranded nucleotides atits 3′ terminus, where the 3′ terminus of the first oligonucleotidestrand and the 5′ terminus of the second oligonucleotide strand form ablunt end, and the second oligonucleotide strand is sufficientlycomplementary to a target HAO1 sequence of SEQ ID NOs: 1921-2304 alongat least 19 nucleotides of the second oligonucleotide strand length toreduce HAO1 mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell.

In one embodiment, the first strand is 15-35 nucleotides in length.

In another embodiment, the second strand is 19-35 nucleotides in length.

In an additional embodiment, the dsNA possesses a duplex region of atleast 25 base pairs; 19-21 base pairs or 21-25 base pairs.

Optionally, the second oligonucleotide strand includes 1-5single-stranded nucleotides at its 3′ terminus.

In one embodiment, the second oligonucleotide strand includes 5-35single-stranded nucleotides at its 3′ terminus.

In another embodiment, the single-stranded nucleotides include modifiednucleotides. Optionally, the single-stranded nucleotides include2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and/or 2′-O—(N-methlycarbamate)modified nucleotides.

In certain embodiments, the single-stranded nucleotides includeribonucleotides. Optionally, the single-stranded nucleotides includedeoxyribonucleotides.

In certain embodiments, the 3′ end of the first strand and the 5′ end ofthe second strand of a dsNA or hybridization complex of the inventionare joined by a polynucleotide sequence that includes ribonucleotides,deoxyribonucleotides or both, optionally the polynucleotide sequenceincludes a tetraloop sequence.

In one embodiment, the first strand is 25-35 nucleotides in length.Optionally, the second strand is 25-35 nucleotides in length.

In one embodiment, the second oligonucleotide strand is complementary totarget HAO1 cDNA sequence GenBank Accession No. NM_017545.2 along atmost 27 nucleotides of the second oligonucleotide strand length.

In another embodiment, starting from the first nucleotide (position 1)at the 3′ terminus of the first oligonucleotide strand, position 1, 2and/or 3 is substituted with a modified nucleotide.

Optionally, the first strand and the 5′ terminus of the second strandform a blunt end.

In one embodiment, the first strand is 25 nucleotides in length and thesecond strand is 27 nucleotides in length.

In another embodiment, starting from the 5′ end of a HAO1 mRNA sequenceof SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNAat a site between positions 9 and 10 of the sequence, thereby reducingHAO1 target mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell.

In one embodiment, the second strand includes a sequence of SEQ ID NOs:385-768. In an additional embodiment, the first strand includes asequence of SEQ ID NOs: 1-384.

In another embodiment, the dsNA of the invention possesses a pair offirst strand/second strand sequences of Table 2.

Optionally, the modified nucleotide residue of the 3′ terminus of thefirst strand is a deoxyribonucleotide, an acyclonucleotide or afluorescent molecule.

In one embodiment, position 1 of the 3′ terminus of the firstoligonucleotide strand is a deoxyribonucleotide.

In another embodiment, the nucleotides of the 1-5 or 5-35single-stranded nucleotides of the 3′ terminus of the second strandcomprise a modified nucleotide.

In one embodiment, the modified nucleotide of the 1-5 or 5-35single-stranded nucleotides of the 3′ terminus of the second strand is a2′-O-methyl ribonucleotide.

In an additional embodiment, all nucleotides of the 1-5 or 5-35single-stranded nucleotides of the 3′ terminus of the second strand aremodified nucleotides.

In another embodiment, the dsNA includes a modified nucleotide.Optionally, the modified nucleotide residue is 2′-O-methyl,2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O—(N-methlycarbamate).

In one embodiment, the 1-5 or 5-35 single-stranded nucleotides of the 3′terminus of the second strand are 1-3 nucleotides in length, optionally1-2 nucleotides in length.

In another embodiment, the 1-5 or 5-35 single-stranded nucleotides ofthe 3′ terminus of the second strand is two nucleotides in length andincludes a 2′-O-methyl modified ribonucleotide.

In one embodiment, the second oligonucleotide strand includes amodification pattern of AS-M1 to AS-M96 and AS-M1* to AS-M96*.

In another embodiment, the first oligonucleotide strand includes amodification pattern of SM1 to SM119.

In an additional embodiment, each of the first and the second strandshas a length which is at least 26 and at most 30 nucleotides.

In one embodiment, the dsNA is cleaved endogenously in the cell byDicer.

In another embodiment, the amount of the nucleic acid sufficient toreduce expression of the target gene is of 1 nanomolar or less, 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, 10 picomolar or less, 5 picomolar or less, 2,picomolar or less and 1 picomolar or less in the environment of thecell.

In one embodiment, the dsNA possesses greater potency than a 21 mersiRNA directed to the identical at least 19 nucleotides of the targetHAO1 mRNA in reducing target HAO1 mRNA expression when assayed in vitroin a mammalian cell at an effective concentration in the environment ofa cell of 1 nanomolar or less.

In an additional embodiment, the nucleic acid or dsNA is sufficientlycomplementary to the target HAO1 mRNA sequence to reduce HAO1 targetmRNA expression by an amount (expressed by %) of at least 10%, at least50%, at least 80-90%, at least 95%, at least 98%, or at least 99% whenthe double stranded nucleic acid is introduced into a mammalian cell.

In one embodiment, the first and second strands are joined by a chemicallinker. In another embodiment, the 3′ terminus of the first strand andthe 5′ terminus of the second strand are joined by a chemical linker.

In an additional embodiment, a nucleotide of the second or first strandis substituted with a modified nucleotide that directs the orientationof Dicer cleavage.

In one embodiment, the nucleic acid, dsNA or hybridization complexpossesses a deoxyribonucleotide, a dideoxyribonucleotide, anacyclonucleotide, a 3′-deoxyadenosine (cordycepin), a3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a2′,3′-dideoxy-3′-thiacytidine (3TC), a2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotideof 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine(3TC) and a monophosphate nucleotide of2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkylribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide,a 2′-fluoro ribonucleotide, or a locked nucleic acid modifiednucleotide.

In another embodiment, the nucleic acid, dsNA or hybridization complexincludes a phosphonate, a phosphorothioate or a phosphotriesterphosphate backbone modification.

In one embodiment, the nucleic acid, dsNA or hybridization complexincludes a morpholino nucleic acid or a peptide nucleic acid (PNA).

In an additional embodiment, the nucleic acid, dsNA or hybridizationcomplex is attached to a dynamic polyconjugate (DPC).

In one embodiment, the nucleic acid, dsNA or hybridization complex isadministered with a DPC, where the dsNA and DPC are optionally notattached.

In another embodiment, the nucleic acid, dsNA or hybridization complexis attached to a GalNAc moiety, a cholesterol and/or a cholesteroltargeting ligand.

Another aspect of the invention provides a composition of the following:

a dsNA possessing first and second nucleic acid strands, where the firststrand is 15-35 nucleotides in length and the second strand of the dsNAis 19-35 nucleotides in length, where the second oligonucleotide strandis sufficiently complementary to a target HAO1 mRNA sequence of SEQ IDNOs: 1921-2304 along at least 19 nucleotides of the secondoligonucleotide strand length to reduce HAO1 target mRNA expression whenthe double stranded nucleic acid is introduced into a mammalian cell;

a dsNA possessing first and second nucleic acid strands, where the firststrand is 15-35 nucleotides in length and the second strand of the dsNAis 19-35 nucleotides in length, where the second oligonucleotide strandis sufficiently complementary to a target HAO1 mRNA sequence of SEQ IDNOs: 1921-2304 along at least 19 nucleotides of the secondoligonucleotide strand length to reduce HAO1 target mRNA expression, andwhere, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs:1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a sitebetween positions 9 and 10 of the sequence, when the double strandednucleic acid is introduced into a mammalian cell;

a dsNA molecule, consisting of: (a) a sense region and an antisenseregion, where the sense region and the antisense region together form aduplex region consisting of 25-35 base pairs and the antisense regionincludes a sequence that is the complement of a sequence of SEQ ID NOs:1921-2304; and (b) from zero to two 3′ overhang regions, where eachoverhang region is six or fewer nucleotides in length, and where,starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs:1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a sitebetween positions 9 and 10 of the sequence, when the double strandednucleic acid is introduced into a mammalian cell;

a dsNA possessing first and second nucleic acid strands and a duplexregion of at least 25 base pairs, where the first strand is 25-34nucleotides in length and the second strand of the dsNA is 26-35nucleotides in length and includes 1-5 single-stranded nucleotides atits 3′ terminus, where the second oligonucleotide strand is sufficientlycomplementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304along at least 19 nucleotides of the second oligonucleotide strandlength to reduce HAO1 target gene expression when the double strandednucleic acid is introduced into a mammalian cell;

a dsNA possessing first and second nucleic acid strands and a duplexregion of at least 25 base pairs, where the first strand is 25-34nucleotides in length and the second strand of the dsNA is 26-35nucleotides in length and includes 1-5 single-stranded nucleotides atits 3′ terminus, where the 3′ terminus of the first oligonucleotidestrand and the 5′ terminus of the second oligonucleotide strand form ablunt end, and the second oligonucleotide strand is sufficientlycomplementary to a target HAO1 sequence of SEQ ID NOs: 1921-2304 alongat least 19 nucleotides of the second oligonucleotide strand length toreduce HAO1 mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell;

a nucleic acid possessing an oligonucleotide strand of 19-35 nucleotidesin length, where the oligonucleotide strand is sufficientlycomplementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304along at least 19 nucleotides of the oligonucleotide strand length toreduce HAO1 target mRNA expression when the nucleic acid is introducedinto a mammalian cell;

a nucleic acid possessing an oligonucleotide strand of 15-35 nucleotidesin length, where the oligonucleotide strand is hybridizable to a targetHAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15nucleotides of the oligonucleotide strand length;

a dsNA possessing first and second nucleic acid strands possessing RNA,where the first strand is 15-35 nucleotides in length and the secondstrand of the dsNA is 19-35 nucleotides in length, where the secondoligonucleotide strand is hybridizable to a target HAO1 mRNA sequence ofSEQ ID NOs: 1921-2304 along at least 15 nucleotides of the secondoligonucleotide strand length;

a dsNA possessing first and second nucleic acid strands possessing RNA,where the first strand is 15-35 nucleotides in length and the secondstrand of the dsNA is at least 35 nucleotides in length and optionallyincludes a sequence of at least 25 nucleotides in length of SEQ ID NOs:385-768, where the second oligonucleotide strand is sufficientlycomplementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304along at least 15 nucleotides of the second oligonucleotide strandlength to reduce HAO1 target mRNA expression when the double strandednucleic acid is introduced into a mammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where the first strand is 25 to 53nucleotide residues in length, where starting from the first nucleotide(position 1) at the 5′ terminus of the first strand, positions 1 to 23of the first strand are ribonucleotides or modified ribonucleotides; thesecond strand is 27 to 53 nucleotide residues in length and includes 23consecutive ribonucleotides or modified ribonucleotides that base pairwith the ribonucleotides or ribonucleotides of positions 1 to 23 of thefirst strand to form a duplex; the 5′ terminus of the first strand andthe 3′ terminus of the second strand form a structure of a blunt end, a1-6 nucleotide 5′ overhang and a 1-6 nucleotide 3′ overhang; the 3′terminus of the first strand and the 5′ terminus of the second strandform a structure of a blunt end, a 1-6 nucleotide 5′ overhang and a 1-6nucleotide 3′ overhang; at least one of positions 24 to the 3′ terminalnucleotide residue of the first strand is a deoxyribonucleotide ormodified ribonucleotide that optionally base pairs with adeoxyribonucleotide of the second strand; and the second strand issufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs:1921-2304 along at least 15 nucleotides of the second oligonucleotidestrand length to reduce HAO1 target mRNA expression when the doublestranded nucleic acid is introduced into a mammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where the second strand is 27 to 53nucleotide residues in length, where starting from the first nucleotide(position 1) at the 5′ terminus of the second strand, positions 1 to 23of the second strand are ribonucleotides or modified ribonucleotides;the first strand is 25 to 53 nucleotide residues in length and includes23 consecutive ribonucleotides or modified ribonucleotides that basepair sufficiently with the ribonucleotides of positions 1 to 23 of thesecond strand to form a duplex; at least one of positions 24 to the 3′terminal nucleotide residue of the second strand is adeoxyribonucleotide or modified ribonucleotide, optionally that basepairs with a deoxyribonucleotide or modified ribonucleotide of the firststrand; and the second strand is sufficiently complementary to a targetHAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15nucleotides of the second oligonucleotide strand length to reduce HAO1target mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each of the 5′ termini has a 5′terminal nucleotide and each of the 3′ termini has a 3′ terminalnucleotide, where the first strand (or the second strand) is 25-30nucleotide residues in length, where starting from the 5′ terminalnucleotide (position 1) positions 1 to 23 of the first strand (or thesecond strand) include at least 8 ribonucleotides; the second strand (orthe first strand) is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, includes at least 8 ribonucleotides inthe positions paired with positions 1-23 of the first strand to form aduplex; where at least the 3′ terminal nucleotide of the second strand(or the first strand) is unpaired with the first strand (or the secondstrand), and up to 6 consecutive 3′ terminal nucleotides are unpairedwith the first strand (or the second strand), thereby forming a 3′single stranded overhang of 1-6 nucleotides; where the 5′ terminus ofthe second strand (or the first strand) includes from 10-30 consecutivenucleotides which are unpaired with the first strand (or the secondstrand), thereby forming a 10-30 nucleotide single stranded 5′ overhang;where at least the first strand (or the second strand) 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of the secondstrand (or first strand) when the first and second strands are alignedfor maximum complementarity, thereby forming a substantially duplexedregion between the first and second strands; and the second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each of the 5′ termini has a 5′terminal nucleotide and each of the 3′ termini has a 3′ terminalnucleotide, where the first strand is 25-35 nucleotide residues inlength, where starting from the 5′ terminal nucleotide (position 1)positions 1 to 25 of the second strand include at least 8ribonucleotides; the second strand is 30-66 nucleotide residues inlength and, starting from the 3′ terminal nucleotide, includes at least8 ribonucleotides in the positions paired with positions 1-25 of thefirst strand to form a duplex; where the 5′ terminus of the secondstrand includes from 5-35 consecutive nucleotides which are unpairedwith the first strand, thereby forming a 5-35 nucleotide single stranded5′ overhang; where at least the first strand 5′ terminal and 3′ terminalnucleotides are base paired with nucleotides of the second strand whenthe first and second strands are aligned for maximum complementarity,thereby forming a substantially duplexed region between the first andsecond strands; and the second strand is sufficiently complementary to atarget HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15nucleotides of the second oligonucleotide strand length to reduce HAO1target mRNA expression when the double stranded nucleic acid isintroduced into a mammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each of the 5′ termini has a 5′terminal nucleotide and each of the 3′ termini has a 3′ terminalnucleotide, where the second strand is 19-30 nucleotide residues inlength and optionally 25-30 nucleotide residues in length, wherestarting from the 5′ terminal nucleotide (position 1) positions 1 to 17(optionally positions 1 to 23) of the second strand include at least 8ribonucleotides; the first strand is 24-66 nucleotide residues in length(optionally 30-66 nucleotide residues in length) and, starting from the3′ terminal nucleotide, includes at least 8 ribonucleotides in thepositions paired with positions 1 to 17 (optionally positions 1 to 23)of the second strand to form a duplex; where the 3′ terminus of thefirst strand and the 5′ terminus of the second strand comprise astructure of a blunt end, a 3′ overhang and a 5′ overhang, optionallywhere the overhang is 1-6 nucleotides in length; where the 5′ terminusof the first strand includes from 5-35 consecutive nucleotides which areunpaired with the second strand, thereby forming a 5-35 nucleotidesingle-stranded 5′ overhang; where at least the second strand 5′terminal and 3′ terminal nucleotides are base paired with nucleotides ofthe first strand when the first and second strands are aligned formaximum complementarity, thereby forming a substantially duplexed regionbetween the first and second strands; and the second strand issufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs:1921-2304 along at least 15 nucleotides of the second oligonucleotidestrand length to reduce HAO1 target mRNA expression when the doublestranded nucleic acid is introduced into a mammalian cell;

a dsNA possessing a first strand and a second strand, where the firststrand and the second strand form a duplex region of 19-25 nucleotidesin length, where the first strand includes a 3′ region that extendsbeyond the first strand-second strand duplex region and includes atetraloop, and the dsNA further includes a discontinuity between the 3′terminus of the first strand and the 5′ terminus of the second strand,and the first or second strand is sufficiently complementary to a targetHAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15nucleotides of the first or second strand length to reduce HAO1 targetmRNA expression when the dsNA is introduced into a mammalian cell;

a dsNA possessing a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each of the 5′ termini has a 5′terminal nucleotide and each of the 3′ termini has a 3′ terminalnucleotide, where the first oligonucleotide strand is 25-53 nucleotidesin length and the second is oligonucleotide strand is 25-53 nucleotidesin length, and where the dsNA is sufficiently highly modified tosubstantially prevent dicer cleavage of the dsNA, optionally where thedsNA is cleaved by non-dicer nucleases to yield one or more 19-23nucleotide strand length dsNAs capable of reducing LDH mRNA expressionin a mammalian cell;

an in vivo hybridization complex within a cell of an exogenous nucleicacid sequence and a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304;and

an in vitro hybridization complex within a cell of an exogenous nucleicacid sequence and a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304.

In one embodiment, a dsNA of the invention possesses a duplex region ofat least 25 base pairs, where the dsNA possesses greater potency than a21 mer siRNA directed to the identical at least 19 nucleotides of thetarget HAO1 mRNA in reducing target HAO1 mRNA expression when assayed invitro in a mammalian cell at an effective concentration in theenvironment of a cell of 1 nanomolar or less.

In another embodiment, the dsNA of the invention has a duplex region ofat least 25 base pairs, where the dsNA possesses greater potency than a21 mer siRNA directed to the identical at least 19 nucleotides of thetarget HAO1 mRNA in reducing target HAO1 mRNA expression when assayed invitro in a mammalian cell at a concentration of 1 nanomolar, 200picomolar, 100 picomolar, 50 picomolar, 20 picomolar, 10 picomolar, 5picomolar, 2, picomolar and 1 picomolar.

In an additional embodiment, the dsNA of the invention possesses four orfewer mismatched nucleic acid residues with respect to the target HAO1mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15or 19 nucleotides of the second oligonucleotide strand when the 15 or 19consecutive nucleotides of the second oligonucleotide are aligned formaximum complementarity with the target HAO1 mRNA sequence.

In another embodiment, the dsNA of the invention possesses three orfewer mismatched nucleic acid residues with respect to the target HAO1mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15or 19 nucleotides of the second oligonucleotide strand when the 15 or 19consecutive nucleotides of the second oligonucleotide are aligned formaximum complementarity with the target HAO1 mRNA sequence.

Optionally, the dsNA of the invention possesses two or fewer mismatchednucleic acid residues with respect to the target HAO1 mRNA sequencealong 15 or 19 consecutive nucleotides of the at least 15 or 19nucleotides of the second oligonucleotide strand when the 15 or 19consecutive nucleotides of the second oligonucleotide are aligned formaximum complementarity with the target HAO1 mRNA sequence.

In certain embodiments, the dsNA of the invention possesses onemismatched nucleic acid residue with respect to the target HAO1 mRNAsequence along 15 or 19 consecutive nucleotides of the at least 15 or 19nucleotides of the second oligonucleotide strand when the 15 or 19consecutive nucleotides of the second oligonucleotide are aligned formaximum complementarity with the target HAO1 mRNA sequence.

In one embodiment, 15 or 19 consecutive nucleotides of the at least 15or 19 nucleotides of the second oligonucleotide strand are completelycomplementary to the target HAO1 mRNA sequence when the 15 or 19consecutive nucleotides of the second oligonucleotide are aligned formaximum complementarity with the target HAO1 mRNA sequence.

Optionally, a nucleic acid, dsNA or hybridization complex of theinvention is isolated.

Another aspect of the invention provides a method for reducingexpression of a target HAO1 gene in a mammalian cell involvingcontacting a mammalian cell in vitro with a nucleic acid, dsNA orhybridization complex of the invention in an amount sufficient to reduceexpression of a target HAO1 mRNA in the cell.

Optionally, target HAO1 mRNA expression is reduced by an amount(expressed by %) of at least 10%, at least 50% and at least 80-90%.

In one embodiment, HAO1 mRNA levels are reduced by an amount (expressedby %) of at least 90% at least 8 days after the cell is contacted withthe dsNA.

In another embodiment, HAO1 mRNA levels are reduced by an amount(expressed by %) of at least 70% at least 10 days after the cell iscontacted with the dsNA.

A further aspect of the invention provides a method for reducingexpression of a target HAO1 mRNA in a mammal possessing administering anucleic acid, dsNA or hybridization complex of the invention to a mammalin an amount sufficient to reduce expression of a target HAO1 mRNA inthe mammal.

In one embodiment, the nucleic acid, dsNA or hybridization complex isformulated in a lipid nanoparticle (LNP).

In another embodiment, the nucleic acid, dsNA or hybridization complexis administered at a dosage of 1 microgram to 5 milligrams per kilogramof the mammal per day, 100 micrograms to 0.5 milligrams per kilogram,0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms perkilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms perkilogram, and 0.1 to 2.5 micrograms per kilogram.

In certain embodiments, the nucleic acid, dsNA or hybridization complexpossesses greater potency than 21 mer siRNAs directed to the identicalat least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1mRNA expression when assayed in vitro in a mammalian cell at aneffective concentration in the environment of a cell of 1 nanomolar orless.

In one embodiment, HAO1 mRNA levels are reduced in a tissue of themammal by an amount (expressed by %) of at least 70% at least 3 daysafter the dsNA is administered to the mammal.

Optionally, the tissue is liver tissue.

In certain embodiments, the administering step involves intravenousinjection, intramuscular injection, intraperitoneal injection, infusion,subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical,oral or inhaled delivery.

Another aspect of the invention provides a method for treating orpreventing a disease or disorder in a subject that involvesadministering to the subject an amount of a nucleic acid, dsNA orhybridization complex of the invention in an amount sufficient to treator prevent the disease or disorder in the subject.

In one embodiment, the disease or disorder is primary hyperoxaluria 1(PH1).

Optionally, the subject is human.

A further aspect of the invention provides a formulation that includesthe nucleic acid, dsNA or hybridization complex of the invention, wherethe nucleic acid, dsNA or hybridization complex is present in an amounteffective to reduce target HAO1 mRNA levels when the nucleic acid, dsNAor hybridization complex is introduced into a mammalian cell in vitro byan amount (expressed by %) of at least 10%, at least 50% and at least80-90%.

In one embodiment, the effective amount is of 1 nanomolar or less, 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, 10 picomolar or less, 5 picomolar or less, 2,picomolar or less and 1 picomolar or less in the environment of thecell.

In another embodiment, the nucleic acid, dsNA or hybridization complexis present in an amount effective to reduce target HAO1 mRNA levels whenthe nucleic acid, dsNA or hybridization complex is introduced into acell of a mammalian subject by an amount (expressed by %) of at least10%, at least 50% and at least 80-90%.

Optionally, the effective amount is a dosage of 1 microgram to 5milligrams per kilogram of the subject per day, 100 micrograms to 0.5milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.

In another embodiment, the nucleic acid, dsNA or hybridization complexpossesses greater potency than an 21 mer siRNA directed to the identicalat least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1mRNA levels when assayed in vitro in a mammalian cell at an effectiveconcentration in the environment of a cell of 1 nanomolar or less.

A further aspect of the invention provides a mammalian cell containingthe nucleic acid, dsNA or hybridization complex of the invention.

Another aspect of the invention provides a pharmaceutical compositionthat includes the nucleic acid, dsNA or hybridization complex of theinvention and a pharmaceutically acceptable carrier.

An additional aspect of the invention provides a kit including thenucleic acid, dsNA or hybridization complex of the invention andinstructions for its use.

A further aspect of the invention provides a composition possessing HAO1inhibitory activity consisting essentially of a nucleic acid, dsNA orhybridization complex of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of exemplary DsiRNA agents of the inventiontargeting a site in the Glycolate Oxidase RNA referred to herein as the“HAO1-1171” target site. UPPER case=unmodified RNA, lower case=DNA,Bold=mismatch base pair nucleotides; arrowheads indicate projected Dicerenzyme cleavage sites; dashed line indicates sense strand (top strand)sequences corresponding to the projected Argonaute 2 (Ago2) cleavagesite within the targeted Glycolate Oxidase sequence.

FIGS. 2A to 2F present primary screen data showing DsiRNA-mediatedknockdown of human Glycolate Oxidase (FIGS. 2A and 2B show results forhuman-mouse common DsiRNAs, while FIGS. 2C and 2D show results for humanunique DsiRNAs) and mouse Glycolate Oxidase (FIGS. 2C and 2D) in human(HeLa) and mouse (Hepa1-6) cells, respectively. Activity in human HeLacells was assessed using a Psi-Check-HsHAO1 plasmid reporter assay,monitoring for knockdown of Renilla luciferase production. For eachDsiRNA tested in mouse cells, two independent qPCR amplicons wereassayed (amplicons “476-611” and “1775-1888”).

FIGS. 3A to 3F show histograms of human and mouse Glycolate Oxidaseinhibitory efficacies observed for indicated DsiRNAs. “P1” indicatesphase 1 (primary screen), while “P2” indicates phase 2. In phase 1,DsiRNAs were tested at 1 nM in the environment of HeLa cells (human cellassays; FIGS. 3A and 3B) or mouse cells (Hepa1-6 cell assays; FIGS. 3Cto 3F). In phase 2, DsiRNAs were tested at 1 nM and 0.1 nM (induplicate) in the environment of HeLa cells or mouse Hepa1-6 cells.Individual bars represent average human (FIGS. 3A and 3B) or mouse(FIGS. 3C to 3F) Glycolate Oxidase levels observed in triplicate, withstandard errors shown. Mouse Glycolate Oxidase levels were normalized toHPRT and Rp123 levels.

FIG. 4 shows a flow chart for initial in vivo mouse experiments, whichincluded glycolate challenge.

FIG. 5 shows HAO1 knockdown in mouse liver for animals administered 0.1mg/kg or 1 mg/kg of indicated HAO1-targeting DsiRNAs formulated in LNP(here, LNP EnCore 2345).

FIG. 6 shows that robust levels of HAO1 mRNA knockdown were observed inliver tissue of mice treated with 1 mg/kg and even 0.1 mg/kg amounts ofHAO1-targeting DsiRNA HAO1-1171, with robust duration of efficaciesobserved (90% or greater levels of knockdown were observed at 120 hourspost-administration for both 1 mg/kg and 0.1 mg/kg animals).

FIGS. 7A to 7E present modification pattern diagrams and histogramsshowing efficacy data for 24 independent HAO1-targeting DsiRNA sequencesacross four different guide (antisense) strand 2′-O-methyl modificationpatterns (“M17”, “M35”, “M48” and “M8”, respectively, as shown in FIG.7A and noting for FIGS. 7B to 7E that modification patterns are recitedas passenger strand modification pattern (here, “M107”)-guide strandmodification pattern, e.g., “M107-M17”, “M107-M35”, “M107-M48” or“M107-M8”) in human HeLa cells tested at 0.1 nM (duplicate assays) and 1nM (FIGS. 7B and 7C) and mouse Hepa1-6 cells for a subset of 8 duplexestested at 0.03 nM, 0.1 nM (duplicate assays) and 1 nM (FIGS. 7D and 7E).

FIGS. 8A to 8E present modification pattern diagrams and histogramsshowing efficacy data for 24 independent HAO1-targeting DsiRNA sequencesacross four different passenger (sense) strand 2′-O-methyl modificationpatterns (“M107”, “M14”, “M24” and “M250”, respectively, as shown inFIG. 8A and noting for FIGS. 8B to 8E that modification patterns arerecited as passenger strand modification pattern-guide (here, “M48”)strand modification pattern, e.g., “M107-M48”, “M14-M48”, “M24-M48” or“M250-M48”) in human HeLa cells tested at 0.1 nM (duplicate assays) and1 nM (FIGS. 8B and 8C) and mouse Hepa1-6 cells for a subset of 8duplexes tested at 0.03 nM, 0.1 nM (duplicate assays) and 1 nM (FIGS. 8Dand 8E).

FIGS. 9A to 9L present modification pattern diagrams and histogramsshowing efficacy data for 24 independent HAO1-targeting DsiRNA sequencesacross indicated passenger (sense) and guide (antisense) strand2′-O-methyl modification patterns (noting that modification patterns arerecited as passenger strand modification pattern-guide strandmodification pattern) in human HeLa cells (FIGS. 9E to 9H) orHEK293-pcDNA HAO1 Cells (FIGS. 9I to 9L), tested at 0.1 nM (duplicateassays) and 1 nM, employing either a Psi-Check-HsHAO1 plasmid reporterassay, monitoring for knockdown of Renilla luciferase production (FIGS.9E to 9H), or HEK293 cells stably transfected with HAO1 (HEK293-pcDNAHAO1 Cells; FIGS. 9I to 9L).

FIGS. 10A to 10O present modification pattern diagrams and histogramsshowing efficacy data for four independent HAO1-targeting DsiRNAsequences across indicated passenger (sense) and guide (antisense)strand 2′-O-methyl modification patterns (noting, as above, thatmodification patterns are recited as: passenger strand modificationpattern-guide strand modification pattern) in HEK293 cells stablytransfected with HAO1 (HEK293-pcDNA HAO1 Cells), assayed at 0.1 nM(duplicate assays) and 1 nM.

FIGS. 11A to 11F present modification patterns and sequences ofHAO1-1171, HAO1-1315, HAO1-1378 and HAO1-1501 DsiRNAs, and associatedinhibitory efficacy results (histograms and IC₅₀ curves), when assayedin HEK293 cells stably transfected with HAO1 (HEK293-pcDNA HAO1 Cells),at 0.1 nM, 0.01 nM and 0.001 nM HAO1-targeting DsiRNA concentrations.

FIGS. 12A to 12F present modification patterns and sequences ofextensively modified HAO1-1171 and HAO1-1376 DsiRNAs as indicated (FIGS.12A to 12D), and associated inhibitory efficacy results (histograms ofFIGS. 12E and 12F), when assayed in HEK293 cells stably transfected withHAO1 (HEK293-pcDNA HAO1 Cells), at 0.1 nM, 0.01 nM and 0.001 nMconcentrations of HAO1-targeting DsiRNAs.

FIGS. 13A to 13L demonstrate the in vivo efficacy—including kinetics andduration of inhibition—of extensively modified forms of LNP-formulatedHAO1-targeting duplexes, both in mice (FIGS. 13A to 13K) and innon-human primates (FIG. 13L).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions that contain nucleicacids, for example double stranded RNA (“dsRNA”), and methods forpreparing them, that are capable of reducing the level and/or expressionof the Glycolate Oxidase (HAO1) gene in vivo or in vitro. One of thestrands of the dsRNA contains a region of nucleotide sequence that has alength that ranges from 19 to 35 nucleotides that can direct thedestruction and/or translational inhibition of the targeted HAO1transcript.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

The present invention features one or more DsiRNA molecules that canmodulate (e.g., inhibit) Glycolate Oxidase expression. The DsiRNAs ofthe invention optionally can be used in combination with modulators ofother genes and/or gene products associated with the maintenance ordevelopment of diseases or disorders associated with Glycolate Oxidasemisregulation (e.g., primary hyperoxaluria 1 (PH1) or other disease ordisorder of the liver, kidneys, etc.). The DsiRNA agents of theinvention modulate Glycolate Oxidase (Hydroxyacid Oxidase 1, HAO1) RNAssuch as those corresponding to the cDNA sequences referred to by GenBankAccession Nos. NM_017545.2 (human HAO1) and NM_010403.2 (mouse HAO1),which are referred to herein generally as “Glycolate Oxidase” or“Hydroxyacid Oxidase 1.”

The below description of the various aspects and embodiments of theinvention is provided with reference to exemplary Glycolate OxidaseRNAs, generally referred to herein as Glycolate Oxidase. However, suchreference is meant to be exemplary only and the various aspects andembodiments of the invention are also directed to alternate GlycolateOxidase RNAs, such as mutant Glycolate Oxidase RNAs or additionalGlycolate Oxidase splice variants. Certain aspects and embodiments arealso directed to other genes involved in Glycolate Oxidase pathways,including genes whose misregulation acts in association with that ofGlycolate Oxidase (or is affected or affects Glycolate Oxidaseregulation) to produce phenotypic effects that may be targeted fortreatment (e.g., glyoxylate and/or oxalate overproduction). DAO and AGTare examples of genes that interact with Glycolate Oxidase. Suchadditional genes, including those of pathways that act in coordinationwith Glycolate Oxidase, can be targeted using dsRNA and the methodsdescribed herein for use of Glycolate Oxidase-targeting dsRNAs. Thus,the inhibition or and the effects of such inhibition, or up-regulationand the effects of such, of the other genes can be performed asdescribed herein.

The term “Glycolate Oxidase” refers to nucleic acid sequences encoding aGlycolate Oxidase protein, peptide, or polypeptide (e.g., GlycolateOxidase transcripts, such as the sequences of Glycolate Oxidase (HAO1)Genbank Accession Nos. NM_017545.2 and NM_010403.2). In certainembodiments, the term “Glycolate Oxidase” is also meant to include otherGlycolate Oxidase encoding sequence, such as other Glycolate Oxidaseisoforms, mutant Glycolate Oxidase genes, splice variants of GlycolateOxidase genes, and Glycolate Oxidase gene polymorphisms. The term“Glycolate Oxidase” is also used to refer to the polypeptide geneproduct of an Glycolate Oxidase gene/transcript, e.g., a GlycolateOxidase protein, peptide, or polypeptide, such as those encoded byGlycolate Oxidase (HAO1) Genbank Accession Nos. NP_060015.1 andNP_034533.1.

As used herein, a “Glycolate Oxidase-associated disease or disorder”refers to a disease or disorder known in the art to be associated withaltered Glycolate Oxidase expression, level and/or activity. Notably, a“Glycolate Oxidase-associated disease or disorder” or “HAO1-associateddisease or disorder” includes diseases or disorders of the kidney,liver, skin, bone, eye and other organs including, but not limited to,primary hyperoxaluria (PH1).

In certain embodiments, dsRNA-mediated inhibition of a Glycolate Oxidasetarget sequence is assessed. In such embodiments, Glycolate Oxidase RNAlevels can be assessed by art-recognized methods (e.g., RT-PCR, Northernblot, expression array, etc.), optionally via comparison of GlycolateOxidase levels in the presence of an anti-Glycolate Oxidase dsRNA of theinvention relative to the absence of such an anti-Glycolate OxidasedsRNA. In certain embodiments, Glycolate Oxidase levels in the presenceof an anti-Glycolate Oxidase dsRNA are compared to those observed in thepresence of vehicle alone, in the presence of a dsRNA directed againstan unrelated target RNA, or in the absence of any treatment.

It is also recognized that levels of Glycolate Oxidase protein can beassessed and that Glycolate Oxidase protein levels are, under differentconditions, either directly or indirectly related to Glycolate OxidaseRNA levels and/or the extent to which a dsRNA inhibits Glycolate Oxidaseexpression, thus art-recognized methods of assessing Glycolate Oxidaseprotein levels (e.g., Western blot, immunoprecipitation, otherantibody-based methods, etc.) can also be employed to examine theinhibitory effect of a dsRNA of the invention.

An anti-Glycolate Oxidase dsRNA of the invention is deemed to possess“Glycolate Oxidase inhibitory activity” if a statistically significantreduction in Glycolate Oxidase RNA (or when the Glycolate Oxidaseprotein is assessed, Glycolate Oxidase protein levels) is seen when ananti-Glycolate Oxidase dsRNA of the invention is administered to asystem (e.g., cell-free in vitro system), cell, tissue or organism, ascompared to a selected control. The distribution of experimental valuesand the number of replicate assays performed will tend to dictate theparameters of what levels of reduction in Glycolate Oxidase RNA (eitheras a % or in absolute terms) is deemed statistically significant (asassessed by standard methods of determining statistical significanceknown in the art). However, in certain embodiments, “Glycolate Oxidaseinhibitory activity” is defined based upon a % or absolute level ofreduction in the level of Glycolate Oxidase in a system, cell, tissue ororganism. For example, in certain embodiments, a dsRNA of the inventionis deemed to possess Glycolate Oxidase inhibitory activity if at least a5% reduction or at least a 10% reduction in Glycolate Oxidase RNA isobserved in the presence of a dsRNA of the invention relative toGlycolate Oxidase levels seen for a suitable control. (For example, invivo Glycolate Oxidase levels in a tissue and/or subject can, in certainembodiments, be deemed to be inhibited by a dsRNA agent of the inventionif, e.g., a 5% or 10% reduction in Glycolate Oxidase levels is observedrelative to a control.) In certain other embodiments, a dsRNA of theinvention is deemed to possess Glycolate Oxidase inhibitory activity ifGlycolate Oxidase RNA levels are observed to be reduced by at least 15%relative to a selected control, by at least 20% relative to a selectedcontrol, by at least 25% relative to a selected control, by at least 30%relative to a selected control, by at least 35% relative to a selectedcontrol, by at least 40% relative to a selected control, by at least 45%relative to a selected control, by at least 50% relative to a selectedcontrol, by at least 55% relative to a selected control, by at least 60%relative to a selected control, by at least 65% relative to a selectedcontrol, by at least 70% relative to a selected control, by at least 75%relative to a selected control, by at least 80% relative to a selectedcontrol, by at least 85% relative to a selected control, by at least 90%relative to a selected control, by at least 95% relative to a selectedcontrol, by at least 96% relative to a selected control, by at least 97%relative to a selected control, by at least 98% relative to a selectedcontrol or by at least 99% relative to a selected control. In someembodiments, complete inhibition of Glycolate Oxidase is required for adsRNA to be deemed to possess Glycolate Oxidase inhibitory activity. Incertain models (e.g., cell culture), a dsRNA is deemed to possessGlycolate Oxidase inhibitory activity if at least a 50% reduction inGlycolate Oxidase levels is observed relative to a suitable control. Incertain other embodiments, a dsRNA is deemed to possess GlycolateOxidase inhibitory activity if at least an 80% reduction in GlycolateOxidase levels is observed relative to a suitable control.

By way of specific example, in Example 2 below, a series of DsiRNAstargeting Glycolate Oxidase were tested for the ability to reduceGlycolate Oxidase mRNA levels in human HeLa or mouse Hepa1-6 cells invitro, at 1 nM concentrations in the environment of such cells and inthe presence of a transfection agent (Lipofectamine™ RNAiMAX,Invitrogen). Within Example 2 below, Glycolate Oxidase inhibitoryactivity was ascribed to those DsiRNAs that were observed to effect atleast a 50% reduction of Glycolate Oxidase mRNA levels under the assayedconditions. It is contemplated that Glycolate Oxidase inhibitoryactivity could also be attributed to a dsRNA under either more or lessstringent conditions than those employed for Example 2 below, even whenthe same or a similar assay and conditions are employed. For example, incertain embodiments, a tested dsRNA of the invention is deemed topossess Glycolate Oxidase inhibitory activity if at least a 10%reduction, at least a 20% reduction, at least a 30% reduction, at leasta 40% reduction, at least a 50% reduction, at least a 60% reduction, atleast a 70% reduction, at least a 75% reduction, at least an 80%reduction, at least an 85% reduction, at least a 90% reduction, or atleast a 95% reduction in Glycolate Oxidase mRNA levels is observed in amammalian cell line in vitro at 1 nM dsRNA concentration or lower in theenvironment of a cell, relative to a suitable control.

Use of other endpoints for determination of whether a double strandedRNA of the invention possesses Glycolate Oxidase inhibitory activity isalso contemplated. Specifically, in one embodiment, in addition to or asan alternative to assessing Glycolate Oxidase mRNA levels, the abilityof a tested dsRNA to reduce Glycolate Oxidase protein levels (e.g., at48 hours after contacting a mammalian cell in vitro or in vivo) isassessed, and a tested dsRNA is deemed to possess Glycolate Oxidaseinhibitory activity if at least a 10% reduction, at least a 20%reduction, at least a 30% reduction, at least a 40% reduction, at leasta 50% reduction, at least a 60% reduction, at least a 70% reduction, atleast a 75% reduction, at least an 80% reduction, at least an 85%reduction, at least a 90% reduction, or at least a 95% reduction inGlycolate Oxidase protein levels is observed in a mammalian cellcontacted with the assayed double stranded RNA in vitro or in vivo,relative to a suitable control. Additional endpoints contemplatedinclude, e.g., assessment of a phenotype associated with reduction ofGlycolate Oxidase levels—e.g., reduction of phenotypes associated withelevated levels of glyoxylate-derived deposits (e.g., deposits ofcalcium oxalate) throughout the body.

Glycolate Oxidase inhibitory activity can also be evaluated over time(duration) and over concentration ranges (potency), with assessment ofwhat constitutes a dsRNA possessing Glycolate Oxidase inhibitoryactivity adjusted in accordance with concentrations administered andduration of time following administration. Thus, in certain embodiments,a dsRNA of the invention is deemed to possess Glycolate Oxidaseinhibitory activity if at least a 50% reduction in Glycolate Oxidaseactivity is observed/persists at a duration of time of 2 hours, 5 hours,10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days,9 days, 10 days or more after administration of the dsRNA to a cell ororganism. In additional embodiments, a dsRNA of the invention is deemedto be a potent Glycolate Oxidase inhibitory agent if Glycolate Oxidaseinihibitory activity (e.g., in certain embodiments, at least 50%inhibition of Glycolate Oxidase) is observed at a concentration of 1 nMor less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less,20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or even 1 pM orless in the environment of a cell, for example, within an in vitro assayfor Glycolate Oxidase inihibitory activity as described herein. Incertain embodiments, a potent Glycolate Oxidase inhibitory dsRNA of theinvention is defined as one that is capable of Glycolate Oxidaseinihibitory activity (e.g., in certain embodiments, at least 20%reduction of Glycolate Oxidase levels) at a formulated concentration of10 mg/kg or less when administered to a subject in an effective deliveryvehicle (e.g., an effective lipid nanoparticle formulation). Preferably,a potent Glycolate Oxidase inhibitory dsRNA of the invention is definedas one that is capable of Glycolate Oxidase inihibitory activity (e.g.,in certain embodiments, at least 50% reduction of Glycolate Oxidaselevels) at a formulated concentration of 5 mg/kg or less whenadministered to a subject in an effective delivery vehicle. Morepreferably, a potent Glycolate Oxidase inhibitory dsRNA of the inventionis defined as one that is capable of Glycolate Oxidase inihibitoryactivity (e.g., in certain embodiments, at least 50% reduction ofGlycolate Oxidase levels) at a formulated concentration of 5 mg/kg orless when administered to a subject in an effective delivery vehicle.Optionally, a potent Glycolate Oxidase inhibitory dsRNA of the inventionis defined as one that is capable of Glycolate Oxidase inhibitoryactivity (e.g., in certain embodiments, at least 50% reduction ofGlycolate Oxidase levels) at a formulated concentration of 2 mg/kg orless, or even 1 mg/kg or less, when administered to a subject in aneffective delivery vehicle.

In certain embodiments, potency of a dsRNA of the invention isdetermined in reference to the number of copies of a dsRNA present inthe cytoplasm of a target cell that are required to achieve a certainlevel of target gene knockdown. For example, in certain embodiments, apotent dsRNA is one capable of causing 50% or greater knockdown of atarget mRNA when present in the cytoplasm of a target cell at a copynumber of 1000 or fewer RISC-loaded antisense strands per cell. Morepreferably, a potent dsRNA is one capable of producing 50% or greaterknockdown of a target mRNA when present in the cytoplasm of a targetcell at a copy number of 500 or fewer RISC-loaded antisense strands percell. Optionally, a potent dsRNA is one capable of producing 50% orgreater knockdown of a target mRNA when present in the cytoplasm of atarget cell at a copy number of 300 or fewer RISC-loaded antisensestrands per cell.

In further embodiments, the potency of a DsiRNA of the invention can bedefined in reference to a 19 to 23 mer dsRNA directed to the same targetsequence within the same target gene. For example, a DsiRNA of theinvention that possesses enhanced potency relative to a corresponding 19to 23 mer dsRNA can be a DsiRNA that reduces a target gene by anadditional 5% or more, an additional 10% or more, an additional 20% ormore, an additional 30% or more, an additional 40% or more, or anadditional 50% or more as compared to a corresponding 19 to 23 merdsRNA, when assayed in an in vitro assay as described herein at asufficiently low concentration to allow for detection of a potencydifference (e.g., transfection concentrations at or below 1 nM in theenvironment of a cell, at or below 100 pM in the environment of a cell,at or below 10 pM in the environment of a cell, at or below 1 nM in theenvironment of a cell, in an in vitro assay as described herein;notably, it is recognized that potency differences can be best detectedvia performance of such assays across a range of concentrations—e.g.,0.1 pM to 10 nM—for purpose of generating a dose-response curve andidentifying an IC₅₀ value associated with a DsiRNA/dsRNA).

Glycolate Oxidase inhibitory levels and/or Glycolate Oxidase levels mayalso be assessed indirectly, e.g., measurement of a reduction of primaryhyperoxaluria 1 phenotype(s) in a subject may be used to assessGlycolate Oxidase levels and/or Glycolate Oxidase inhibitory efficacy ofa double-stranded nucleic acid of the instant invention.

In certain embodiments, the phrase “consists essentially of” is used inreference to the anti-Glycolate Oxidase dsRNAs of the invention. In somesuch embodiments, “consists essentially of” refers to a composition thatcomprises a dsRNA of the invention which possesses at least a certainlevel of Glycolate Oxidase inhibitory activity (e.g., at least 50%Glycolate Oxidase inhibitory activity) and that also comprises one ormore additional components and/or modifications that do notsignificantly impact the Glycolate Oxidase inhibitory activity of thedsRNA. For example, in certain embodiments, a composition “consistsessentially of” a dsRNA of the invention where modifications of thedsRNA of the invention and/or dsRNA-associated components of thecomposition do not alter the Glycolate Oxidase inhibitory activity(optionally including potency or duration of Glycolate Oxidaseinhibitory activity) by greater than 3%, greater than 5%, greater than10%, greater than 15%, greater than 20%, greater than 25%, greater than30%, greater than 35%, greater than 40%, greater than 45%, or greaterthan 50% relative to the dsRNA of the invention in isolation. In certainembodiments, a composition is deemed to consist essentially of a dsRNAof the invention even if more dramatic reduction of Glycolate Oxidaseinhibitory activity (e.g., 80% reduction, 90% reduction, etc. inefficacy, duration and/or potency) occurs in the presence of additionalcomponents or modifications, yet where Glycolate Oxidase inhibitoryactivity is not significantly elevated (e.g., observed levels ofGlycolate Oxidase inhibitory activity are within 10% those observed forthe isolated dsRNA of the invention) in the presence of additionalcomponents and/or modifications.

As used herein, the term “nucleic acid” refers to deoxyribonucleotides,ribonucleotides, or modified nucleotides, and polymers thereof insingle- or double-stranded form. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs) and unlocked nucleic acids (UNAs; see, e.g., Jensen et al.Nucleic Acids Symposium Series 52: 133-4), and derivatives thereof.

As used herein, “nucleotide” is used as recognized in the art to includethose with natural bases (standard), and modified bases well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Nucleotides generally comprise a base, sugarand a phosphate group. The nucleotides can be unmodified or modified atthe sugar, phosphate and/or base moiety, (also referred tointerchangeably as nucleotide analogs, modified nucleotides, non-naturalnucleotides, non-standard nucleotides and other; see, e.g., Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, hypoxanthine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

As used herein, “modified nucleotide” refers to a nucleotide that hasone or more modifications to the nucleoside, the nucleobase, pentosering, or phosphate group. For example, modified nucleotides excluderibonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate. Modifications include those naturallyoccurring that result from modification by enzymes that modifynucleotides, such as methyltransferases. Modified nucleotides alsoinclude synthetic or non-naturally occurring nucleotides. Synthetic ornon-naturally occurring modifications in nucleotides include those with2′ modifications, e.g., 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge,4′-(CH₂)₂—O-2′-bridge, 2′-LNA or other bicyclic or “bridged” nucleosideanalog, and 2′-O—(N-methylcarbamate) or those comprising base analogs.In connection with 2′-modified nucleotides as described for the presentdisclosure, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can bemodified or unmodified. Such modified groups are described, e.g., inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878. “Modified nucleotides” of the instant invention canalso include nucleotide analogs as described above.

In reference to the nucleic acid molecules of the present disclosure,modifications may exist upon these agents in patterns on one or bothstrands of the double stranded ribonucleic acid (dsRNA). As used herein,“alternating positions” refers to a pattern where every other nucleotideis a modified nucleotide or there is an unmodified nucleotide (e.g., anunmodified ribonucleotide) between every modified nucleotide over adefined length of a strand of the dsRNA (e.g., 5′-MNMNMN-3′;3′-MNMNMN-5′; where M is a modified nucleotide and N is an unmodifiednucleotide). The modification pattern starts from the first nucleotideposition at either the 5′ or 3′ terminus according to a positionnumbering convention, e.g., as described herein (in certain embodiments,position 1 is designated in reference to the terminal residue of astrand following a projected Dicer cleavage event of a DsiRNA agent ofthe invention; thus, position 1 does not always constitute a 3′ terminalor 5′ terminal residue of a pre-processed agent of the invention). Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but in certain embodiments includes at least4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7modified nucleotides, respectively. As used herein, “alternating pairsof positions” refers to a pattern where two consecutive modifiednucleotides are separated by two consecutive unmodified nucleotides overa defined length of a strand of the dsRNA (e.g., 5′-MMNNMMNNMMNN-3′;3′-MMNNMMNNMMNN-5′; where M is a modified nucleotide and N is anunmodified nucleotide). The modification pattern starts from the firstnucleotide position at either the 5′ or 3′ terminus according to aposition numbering convention such as those described herein. Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but preferably includes at least 8, 12, 16,20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14modified nucleotides, respectively. It is emphasized that the abovemodification patterns are exemplary and are not intended as limitationson the scope of the invention.

As used herein, “base analog” refers to a heterocyclic moiety which islocated at the 1′ position of a nucleotide sugar moiety in a modifiednucleotide that can be incorporated into a nucleic acid duplex (or theequivalent position in a nucleotide sugar moiety substitution that canbe incorporated into a nucleic acid duplex). In the dsRNAs of theinvention, a base analog is generally either a purine or pyrimidine baseexcluding the common bases guanine (G), cytosine (C), adenine (A),thymine (T), and uracil (U). Base analogs can duplex with other bases orbase analogs in dsRNAs. Base analogs include those useful in thecompounds and methods of the invention, e.g., those disclosed in U.S.Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent PublicationNo. 20080213891 to Manoharan, which are herein incorporated byreference. Non-limiting examples of bases include hypoxanthine (I),xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K),3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione)(P), iso-cytosine (iso-C), iso-guanine (iso-G),1-β-D-ribofuranosyl-(5-nitroindole),1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine,4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) andpyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S),2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole,4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl,7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl,tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer etal., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic AcidsResearch, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc.,119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324(1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Moraleset al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J.Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem.,63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci.,94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans.,1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1:1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632(2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri etal., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No.6,218,108.). Base analogs may also be a universal base.

As used herein, “universal base” refers to a heterocyclic moiety locatedat the 1′ position of a nucleotide sugar moiety in a modifiednucleotide, or the equivalent position in a nucleotide sugar moietysubstitution, that, when present in a nucleic acid duplex, can bepositioned opposite more than one type of base without altering thedouble helical structure (e.g., the structure of the phosphatebackbone). Additionally, the universal base does not destroy the abilityof the single stranded nucleic acid in which it resides to duplex to atarget nucleic acid. The ability of a single stranded nucleic acidcontaining a universal base to duplex a target nucleic can be assayed bymethods apparent to one in the art (e.g., UV absorbance, circulardichroism, gel shift, single stranded nuclease sensitivity, etc.).Additionally, conditions under which duplex formation is observed may bevaried to determine duplex stability or formation, e.g., temperature, asmelting temperature (Tm) correlates with the stability of nucleic acidduplexes. Compared to a reference single stranded nucleic acid that isexactly complementary to a target nucleic acid, the single strandednucleic acid containing a universal base forms a duplex with the targetnucleic acid that has a lower Tm than a duplex formed with thecomplementary nucleic acid. However, compared to a reference singlestranded nucleic acid in which the universal base has been replaced witha base to generate a single mismatch, the single stranded nucleic acidcontaining the universal base forms a duplex with the target nucleicacid that has a higher Tm than a duplex formed with the nucleic acidhaving the mismatched base.

Some universal bases are capable of base pairing by forming hydrogenbonds between the universal base and all of the bases guanine (G),cytosine (C), adenine (A), thymine (T), and uracil (U) under base pairforming conditions. A universal base is not a base that forms a basepair with only one single complementary base. In a duplex, a universalbase may form no hydrogen bonds, one hydrogen bond, or more than onehydrogen bond with each of G, C, A, T, and U opposite to it on theopposite strand of a duplex. Preferably, the universal bases does notinteract with the base opposite to it on the opposite strand of aduplex. In a duplex, base pairing between a universal base occurswithout altering the double helical structure of the phosphate backbone.A universal base may also interact with bases in adjacent nucleotides onthe same nucleic acid strand by stacking interactions. Such stackinginteractions stabilize the duplex, especially in situations where theuniversal base does not form any hydrogen bonds with the base positionedopposite to it on the opposite strand of the duplex. Non-limitingexamples of universal-binding nucleotides include inosine,1-β-D-ribofuranosyl-5-nitroindole, and/or1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazolenucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindoleas universal bases in primers for DNA sequencing and PCR. Nucleic AcidsRes. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as anuniversal base analogue. Nucleic Acids Res. 1994 Oct. 11;22(20):4039-43).

As used herein, “loop” refers to a structure formed by a single strandof a nucleic acid, in which complementary regions that flank aparticular single stranded nucleotide region hybridize in a way that thesingle stranded nucleotide region between the complementary regions isexcluded from duplex formation or Watson-Crick base pairing. A loop is asingle stranded nucleotide region of any length. Examples of loopsinclude the unpaired nucleotides present in such structures as hairpins,stem loops, or extended loops.

As used herein, “extended loop” in the context of a dsRNA refers to asingle stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 basepairs or duplexes flanking the loop. In an extended loop, nucleotidesthat flank the loop on the 5′ side form a duplex with nucleotides thatflank the loop on the 3′ side. An extended loop may form a hairpin orstem loop.

As used herein, “tetraloop” in the context of a dsRNA refers to a loop(a single stranded region) consisting of four nucleotides that forms astable secondary structure that contributes to the stability of adjacentWatson-Crick hybridized nucleotides. Without being limited to theory, atetraloop may stabilize an adjacent Watson-Crick base pair by stackinginteractions. In addition, interactions among the four nucleotides in atetraloop include but are not limited to non-Watson-Crick base pairing,stacking interactions, hydrogen bonding, and contact interactions(Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi,Science 1991 Jul. 12; 253(5016):191-4). A tetraloop confers an increasein the melting temperature (Tm) of an adjacent duplex that is higherthan expected from a simple model loop sequence consisting of fourrandom bases. For example, a tetraloop can confer a melting temperatureof at least 55° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex ofat least 2 base pairs in length. A tetraloop may containribonucleotides, deoxyribonucleotides, modified nucleotides, andcombinations thereof. Examples of RNA tetraloops include the UNCG familyof tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA),and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci USA. 1990November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11;19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family oftetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, thed(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, thed(TNCG) family of tetraloops (e.g., d(TTCG)). (Nakano et al.Biochemistry, 41 (48), 14281-14292, 2002; SHINJI et al. Nippon KagakkaiKoen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

As used herein, the term “siRNA” refers to a double stranded nucleicacid in which each strand comprises RNA, RNA analog(s) or RNA and DNA.The siRNA comprises between 19 and 23 nucleotides or comprises 21nucleotides. The siRNA typically has 2 bp overhangs on the 3′ ends ofeach strand such that the duplex region in the siRNA comprises 17-21nucleotides, or 19 nucleotides. Typically, the antisense strand of thesiRNA is sufficiently complementary with the target sequence of theGlycolate Oxidase gene/RNA.

In certain embodiments, an anti-Glycolate Oxidase DsiRNA of the instantinvention possesses strand lengths of at least 25 nucleotides.Accordingly, in certain embodiments, an anti-Glycolate Oxidase DsiRNAcontains one oligonucleotide sequence, a first sequence, that is atleast 25 nucleotides in length and no longer than 35 or up to 50 or morenucleotides. This sequence of RNA can be between 26 and 35, 26 and 34,26 and 33, 26 and 32, 26 and 31, 26 and 30, and 26 and 29 nucleotides inlength. This sequence can be 27 or 28 nucleotides in length or 27nucleotides in length. The second sequence of the DsiRNA agent can be asequence that anneals to the first sequence under biological conditions,such as within the cytoplasm of a eukaryotic cell. Generally, the secondoligonucleotide sequence will have at least 19 complementary base pairswith the first oligonucleotide sequence, more typically the secondoligonucleotide sequence will have 21 or more complementary base pairs,or 25 or more complementary base pairs with the first oligonucleotidesequence. In one embodiment, the second sequence is the same length asthe first sequence, and the DsiRNA agent is blunt ended. In anotherembodiment, the ends of the DsiRNA agent have one or more overhangs.

In certain embodiments, the first and second oligonucleotide sequencesof the DsiRNA agent exist on separate oligonucleotide strands that canbe and typically are chemically synthesized. In some embodiments, bothstrands are between 26 and 35 nucleotides in length. In otherembodiments, both strands are between 25 and 30 or 26 and 30 nucleotidesin length. In one embodiment, both strands are 27 nucleotides in length,are completely complementary and have blunt ends. In certain embodimentsof the instant invention, the first and second sequences of ananti-Glycolate Oxidase DsiRNA exist on separate RNA oligonucleotides(strands). In one embodiment, one or both oligonucleotide strands arecapable of serving as a substrate for Dicer. In other embodiments, atleast one modification is present that promotes Dicer to bind to thedouble-stranded RNA structure in an orientation that maximizes thedouble-stranded RNA structure's effectiveness in inhibiting geneexpression. In certain embodiments of the instant invention, theanti-Glycolate Oxidase DsiRNA agent is comprised of two oligonucleotidestrands of differing lengths, with the anti-Glycolate Oxidase DsiRNApossessing a blunt end at the 3′ terminus of a first strand (sensestrand) and a 3′ overhang at the 3′ terminus of a second strand(antisense strand). The DsiRNA can also contain one or moredeoxyribonucleic acid (DNA) base substitutions.

Suitable DsiRNA compositions that contain two separate oligonucleotidescan be chemically linked outside their annealing region by chemicallinking groups. Many suitable chemical linking groups are known in theart and can be used. Suitable groups will not block Dicer activity onthe DsiRNA and will not interfere with the directed destruction of theRNA transcribed from the target gene. Alternatively, the two separateoligonucleotides can be linked by a third oligonucleotide such that ahairpin structure is produced upon annealing of the two oligonucleotidesmaking up the DsiRNA composition. The hairpin structure will not blockDicer activity on the DsiRNA and will not interfere with the directeddestruction of the target RNA.

The dsRNA molecule can be designed such that every residue of theantisense strand is complementary to a residue in the target molecule.Alternatively, substitutions can be made within the molecule to increasestability and/or enhance processing activity of said molecule.Substitutions can be made within the strand or can be made to residuesat the ends of the strand. In certain embodiments, substitutions and/ormodifications are made at specific residues within a DsiRNA agent. Suchsubstitutions and/or modifications can include, e.g.,deoxy-modifications at one or more residues of positions 1, 2 and 3 whennumbering from the 3′ terminal position of the sense strand of a DsiRNAagent; and introduction of 2′-O-alkyl (e.g., 2′-O-methyl) modificationsat the 3′ terminal residue of the antisense strand of DsiRNA agents,with such modifications also being performed at overhang positions ofthe 3′ portion of the antisense strand and at alternating residues ofthe antisense strand of the DsiRNA that are included within the regionof a DsiRNA agent that is processed to form an active siRNA agent. Thepreceding modifications are offered as exemplary, and are not intendedto be limiting in any manner. Further consideration of the structure ofpreferred DsiRNA agents, including further description of themodifications and substitutions that can be performed upon theanti-Glycolate Oxidase DsiRNA agents of the instant invention, can befound below.

Where a first sequence is referred to as “substantially complementary”with respect to a second sequence herein, the two sequences can be fullycomplementary, or they may form one or more, but generally not more than4, 3 or 2 mismatched base pairs upon hybridization, while retaining theability to hybridize under the conditions most relevant to theirultimate application. However, where two oligonucleotides are designedto form, upon hybridization, one or more single stranded overhangs, suchoverhangs shall not be regarded as mismatches with regard to thedetermination of complementarity. For example, a dsRNA comprising oneoligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises asequence of 21 nucleotides that is fully complementary to the shorteroligonucleotide, may yet be referred to as “fully complementary” for thepurposes of the invention.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary, as definedabove, nucleic acid strands. The two strands forming the duplexstructure may be different portions of one larger RNA molecule, or theymay be separate RNA molecules. Where separate RNA molecules, such dsRNAare often referred to as siRNA (“short interfering RNA”) or DsiRNA(“Dicer substrate siRNAs”). Where the two strands are part of one largermolecule, and therefore are connected by an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′ end of therespective other strand forming the duplex structure, the connecting RNAchain is referred to as a “hairpin loop”, “short hairpin RNA” or“shRNA”. Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker”. TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, a dsRNA may comprise one ormore nucleotide overhangs. In addition, as used herein, “dsRNA” mayinclude chemical modifications to ribonucleotides, internucleosidelinkages, end-groups, caps, and conjugated moieties, includingsubstantial modifications at multiple nucleotides and including alltypes of modifications disclosed herein or known in the art. Any suchmodifications, as used in an siRNA- or DsiRNA-type molecule, areencompassed by “dsRNA” for the purposes of this specification andclaims.

The phrase “duplex region” refers to the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or other manner thatallows for a duplex between oligonucleotide strands that arecomplementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 bases oneach strand are complementary or substantially complementary, such thatthe “duplex region” consists of 19 base pairs. The remaining base pairsmay, for example, exist as 5′ and 3′ overhangs. Further, within theduplex region, 100% complementarity is not required; substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to complementarity between the strands such thatthey are capable of annealing under biological conditions. Techniques toempirically determine if two strands are capable of annealing underbiological conditions are well know in the art. Alternatively, twostrands can be synthesized and added together under biologicalconditions to determine if they anneal to one another.

Single-stranded nucleic acids that base pair over a number of bases aresaid to “hybridize.” Hybridization is typically determined underphysiological or biologically relevant conditions (e.g., intracellular:pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion).Hybridization conditions generally contain a monovalent cation andbiologically acceptable buffer and may or may not contain a divalentcation, complex anions, e.g. gluconate from potassium gluconate,uncharged species such as sucrose, and inert polymers to reduce theactivity of water in the sample, e.g. PEG. Such conditions includeconditions under which base pairs can form.

Hybridization is measured by the temperature required to dissociatesingle stranded nucleic acids forming a duplex, i.e., (the meltingtemperature; Tm). Hybridization conditions are also conditions underwhich base pairs can form. Various conditions of stringency can be usedto determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger(1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507). Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Forexample, a hybridization determination buffer is shown in Table 1.

TABLE 1 To make 50 mL final conc. Vender Cat# Lot# m.w./Stock solutionNaCl 100 mM Sigma S-5150 41K8934 5M 1 mL KCl 80 mM Sigma P-9541 70K000274.55 0.298 g MgCl₂ 8 mM Sigma M-1028 120K8933 1M 0.4 mL sucrose 2% w/vFisher BP220- 907105 342.3 1 g 212 Tris-HCl 16 mM Fisher BP1757- 124191M 0.8 mL 500 NaH₂PO₄ 1 mM Sigma S-3193 52H- 120.0 0.006 g 029515 EDTA0.02 mM Sigma E-7889 110K89271   0.5M 2 μL H₂O Sigma W-4502 51K2359 to50 mL pH = 7.0 adjust with at 20° C. HCl

Useful variations on hybridization conditions will be readily apparentto those skilled in the art. Hybridization techniques are well known tothose skilled in the art and are described, for example, in Benton andDavis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001); Berger and Kimmel(Antisense to Molecular Cloning Techniques, 1987, Academic Press, NewYork); and Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York.

As used herein, “oligonucleotide strand” is a single stranded nucleicacid molecule. An oligonucleotide may comprise ribonucleotides,deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2′modifications, synthetic base analogs, etc.) or combinations thereof.Such modified oligonucleotides can be preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases.

As used herein, the term “ribonucleotide” encompasses natural andsynthetic, unmodified and modified ribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between ribonucleotides in the oligonucleotide. As used herein,the term “ribonucleotide” specifically excludes a deoxyribonucleotide,which is a nucleotide possessing a single proton group at the 2′ ribosering position.

As used herein, the term “deoxyribonucleotide” encompasses natural andsynthetic, unmodified and modified deoxyribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between deoxyribonucleotide in the oligonucleotide. As usedherein, the term “deoxyribonucleotide” also includes a modifiedribonucleotide that does not permit Dicer cleavage of a dsRNA agent,e.g., a 2′-O-methyl ribonucleotide, a phosphorothioate-modifiedribonucleotide residue, etc., that does not permit Dicer cleavage tooccur at a bond of such a residue.

As used herein, the term “PS-NA” refers to a phosphorothioate-modifiednucleotide residue. The term “PS-NA” therefore encompasses bothphosphorothioate-modified ribonucleotides (“PS-RNAs”) andphosphorothioate-modified deoxyribonucleotides (“PS-DNAs”).

As used herein, “Dicer” refers to an endoribonuclease in the RNase IIIfamily that cleaves a dsRNA or dsRNA-containing molecule, e.g.,double-stranded RNA (dsRNA) or pre-microRNA (miRNA), intodouble-stranded nucleic acid fragments 19-25 nucleotides long, usuallywith a two-base overhang on the 3′ end. With respect to certain dsRNAsof the invention (e.g., “DsiRNAs”), the duplex formed by a dsRNA regionof an agent of the invention is recognized by Dicer and is a Dicersubstrate on at least one strand of the duplex. Dicer catalyzes thefirst step in the RNA interference pathway, which consequently resultsin the degradation of a target RNA. The protein sequence of human Diceris provided at the NCBI database under accession number NP_085124,hereby incorporated by reference.

Dicer “cleavage” can be determined as follows (e.g., see Collingwood etal., Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNAduplexes (100 pmol) are incubated in 20 μL of 20 mM Tris pH 8.0, 200 mMNaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer(Stratagene, La Jolla, Calif.) at 37° C. for 18-24 hours. Samples aredesalted using a Performa SR 96-well plate (Edge Biosystems,Gaithersburg, Md.). Electrospray-ionization liquid chromatography massspectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment withDicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hailet al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur datasystem, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources, Auburn, Calif.). In this assay, Dicer cleavage occurswhere at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, oreven 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA,preferably 26-30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bpdsRNA, preferably, 21-23 bp dsRNA).

As used herein, “Dicer cleavage site” refers to the sites at which Dicercleaves a dsRNA (e.g., the dsRNA region of a DsiRNA agent of theinvention). Dicer contains two RNase III domains which typically cleaveboth the sense and antisense strands of a dsRNA. The average distancebetween the RNase III domains and the PAZ domain determines the lengthof the short double-stranded nucleic acid fragments it produces and thisdistance can vary (Macrae et al. (2006) Science 311: 195-8). As shown inFIG. 1, Dicer is projected to cleave certain double-stranded ribonucleicacids of the instant invention that possess an antisense strand having a2 nucleotide 3′ overhang at a site between the 21^(st) and 22^(nd)nucleotides removed from the 3′ terminus of the antisense strand, and ata corresponding site between the 21^(st) and 22^(nd) nucleotides removedfrom the 5′ terminus of the sense strand. The projected and/or prevalentDicer cleavage site(s) for dsRNA molecules distinct from those depictedin FIG. 1 may be similarly identified via art-recognized methods,including those described in Macrae et al. While the Dicer cleavageevents depicted in FIG. 1 generate 21 nucleotide siRNAs, it is notedthat Dicer cleavage of a dsRNA (e.g., DsiRNA) can result in generationof Dicer-processed siRNA lengths of 19 to 23 nucleotides in length.Indeed, in certain embodiments, a double-stranded DNA region may beincluded within a dsRNA for purpose of directing prevalent Dicerexcision of a typically non-preferred 19 mer or 20 mer siRNA, ratherthan a 21 mer.

As used herein, “overhang” refers to unpaired nucleotides, in thecontext of a duplex having one or more free ends at the 5′ terminus or3′ terminus of a dsRNA. In certain embodiments, the overhang is a 3′ or5′ overhang on the antisense strand or sense strand. In someembodiments, the overhang is a 3′ overhang having a length of betweenone and six nucleotides, optionally one to five, one to four, one tothree, one to two, two to six, two to five, two to four, two to three,three to six, three to five, three to four, four to six, four to five,five to six nucleotides, or one, two, three, four, five or sixnucleotides. “Blunt” or “blunt end” means that there are no unpairednucleotides at that end of the dsRNA, i.e., no nucleotide overhang. Forclarity, chemical caps or non-nucleotide chemical moieties conjugated tothe 3′ end or 5′ end of an siRNA are not considered in determiningwhether an siRNA has an overhang or is blunt ended. In certainembodiments, the invention provides a dsRNA molecule for inhibiting theexpression of the Glycolate Oxidase target gene in a cell or mammal,wherein the dsRNA comprises an antisense strand comprising a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of the Glycolate Oxidase target gene, andwherein the region of complementarity is less than 35 nucleotides inlength, optionally 19-24 nucleotides in length or 25-30 nucleotides inlength, and wherein the dsRNA, upon contact with a cell expressing theGlycolate Oxidase target gene, inhibits the expression of the GlycolateOxidase target gene by at least 10%, 25%, or 40%.

A dsRNA of the invention comprises two RNA strands that are sufficientlycomplementary to hybridize to form a duplex structure. One strand of thedsRNA (the antisense strand) comprises a region of complementarity thatis substantially complementary, and generally fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the Glycolate Oxidase target gene, the other strand (thesense strand) comprises a region which is complementary to the antisensestrand, such that the two strands hybridize and form a duplex structurewhen combined under suitable conditions. Generally, the duplex structureis between 15 and 80, or between 15 and 53, or between 15 and 35,optionally between 25 and 30, between 26 and 30, between 18 and 25,between 19 and 24, or between 19 and 21 base pairs in length. Similarly,the region of complementarity to the target sequence is between 15 and35, optionally between 18 and 30, between 25 and 30, between 19 and 24,or between 19 and 21 nucleotides in length. The dsRNA of the inventionmay further comprise one or more single-stranded nucleotide overhang(s).It has been identified that dsRNAs comprising duplex structures ofbetween 15 and 35 base pairs in length can be effective in inducing RNAinterference, including DsiRNAs (generally of at least 25 base pairs inlength) and siRNAs (in certain embodiments, duplex structures of siRNAsare between 20 and 23, and optionally, specifically 21 base pairs(Elbashir et al., EMBO 20: 6877-6888)). It has also been identified thatdsRNAs possessing duplexes shorter than 20 base pairs can be effectiveas well (e.g., 15, 16, 17, 18 or 19 base pair duplexes). In certainembodiments, the dsRNAs of the invention can comprise at least onestrand of a length of 19 nucleotides or more. In certain embodiments, itcan be reasonably expected that shorter dsRNAs comprising a sequencecomplementary to one of the sequences of Tables 4, 6, 8 or 10, minusonly a few nucleotides on one or both ends may be similarly effective ascompared to the dsRNAs described above and in Tables 2, 3, 5, 7 and 9.Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18,19, 20, or more contiguous nucleotides sufficiently complementary to oneof the sequences of Tables 4, 6, 8 or 10, and differing in their abilityto inhibit the expression of the Glycolate Oxidase target gene in anassay as described herein by not more than 5, 10, 15, 20, 25, or 30%inhibition from a dsRNA comprising the full sequence, are contemplatedby the invention. In one embodiment, at least one end of the dsRNA has asingle-stranded nucleotide overhang of 1 to 5, optionally 1 to 4, incertain embodiments, 1 or 2 nucleotides. Certain dsRNA structures havingat least one nucleotide overhang possess superior inhibitory propertiesas compared to counterparts possessing base-paired blunt ends at bothends of the dsRNA molecule.

As used herein, the term “RNA processing” refers to processingactivities performed by components of the siRNA, miRNA or RNase Hpathways (e.g., Drosha, Dicer, Argonaute2 or other RISCendoribonucleases, and RNaseH), which are described in greater detailbelow (see “RNA Processing” section below). The term is explicitlydistinguished from the post-transcriptional processes of 5′ capping ofRNA and degradation of RNA via non-RISC- or non-RNase H-mediatedprocesses. Such “degradation” of an RNA can take several forms, e.g.deadenylation (removal of a 3′ poly(A) tail), and/or nuclease digestionof part or all of the body of the RNA by one or more of several endo- orexo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3,4/5), oligonucleotidase, etc.).

By “homologous sequence” is meant a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of thedsRNA agents of the instant invention contemplates the possibility ofusing such dsRNA agents not only against target RNAs of GlycolateOxidase possessing perfect complementarity with the presently describeddsRNA agents, but also against target Glycolate Oxidase RNAs possessingsequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.complementary to said dsRNA agents. Similarly, it is contemplated thatthe presently described dsRNA agents of the instant invention might bereadily altered by the skilled artisan to enhance the extent ofcomplementarity between said dsRNA agents and a target Glycolate OxidaseRNA, e.g., of a specific allelic variant of Glycolate Oxidase (e.g., anallele of enhanced therapeutic interest). Indeed, dsRNA agent sequenceswith insertions, deletions, and single point mutations relative to thetarget Glycolate Oxidase sequence can also be effective for inhibition.Alternatively, dsRNA agent sequences with nucleotide analogsubstitutions or insertions can be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for comparison purposes (e.g., gaps can be introduced in thefirst sequence or second sequence for optimal alignment). Thenucleotides (or amino acid residues) at corresponding nucleotide (oramino acid) positions are then compared. When a position in the firstsequence is occupied by the same residue as the corresponding positionin the second sequence, then the molecules are identical at thatposition. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e., %homology=# of identical positions/total # of positions×100), optionallypenalizing the score for the number of gaps introduced and/or length ofgaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, a gapped alignment, the alignment is optimized byintroducing appropriate gaps, and percent identity is determined overthe length of the aligned sequences (i.e., a gapped alignment). Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, a global alignment thealignment is optimized by introducing appropriate gaps, and percentidentity is determined over the entire length of the sequences aligned.(i.e., a global alignment). A preferred, non-limiting example of amathematical algorithm utilized for the global comparison of sequencesis the algorithm of Myers and Miller, CABIOS (1989). Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the dsRNA antisense strand and theportion of the Glycolate Oxidase RNA sequence is preferred.Alternatively, the dsRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith a portion of the Glycolate Oxidase RNA (e.g., 400 mM NaCl, 40 mMPIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours;followed by washing). Additional preferred hybridization conditionsinclude hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50%formamide followed by washing at 70° C. in 0.3×SSC or hybridization at70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at67° C. in 1×SSC. The hybridization temperature for hybrids anticipatedto be less than 50 base pairs in length should be 5-10° C. less than themelting temperature (Tm) of the hybrid, where Tm is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in thehybrid, and [Na+] is the concentration of sodium ions in thehybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examples ofstringency conditions for polynucleotide hybridization are provided inSambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11, and Current Protocols in MolecularBiology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4. The length of the identical nucleotidesequences may be at least 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a dsRNA moleculehaving complementarity to an antisense region of the dsRNA molecule. Inaddition, the sense region of a dsRNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a dsRNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a dsRNA molecule comprises a nucleic acidsequence having complementarity to a sense region of the dsRNA molecule.

As used herein, “antisense strand” refers to a single stranded nucleicacid molecule which has a sequence complementary to that of a targetRNA. When the antisense strand contains modified nucleotides with baseanalogs, it is not necessarily complementary over its entire length, butmust at least hybridize with a target RNA.

As used herein, “sense strand” refers to a single stranded nucleic acidmolecule which has a sequence complementary to that of an antisensestrand. When the antisense strand contains modified nucleotides withbase analogs, the sense strand need not be complementary over the entirelength of the antisense strand, but must at least duplex with theantisense strand.

As used herein, “guide strand” refers to a single stranded nucleic acidmolecule of a dsRNA or dsRNA-containing molecule, which has a sequencesufficiently complementary to that of a target RNA to result in RNAinterference. After cleavage of the dsRNA or dsRNA-containing moleculeby Dicer, a fragment of the guide strand remains associated with RISC,binds a target RNA as a component of the RISC complex, and promotescleavage of a target RNA by RISC. As used herein, the guide strand doesnot necessarily refer to a continuous single stranded nucleic acid andmay comprise a discontinuity, preferably at a site that is cleaved byDicer. A guide strand is an antisense strand.

As used herein, “passenger strand” refers to an oligonucleotide strandof a dsRNA or dsRNA-containing molecule, which has a sequence that iscomplementary to that of the guide strand. As used herein, the passengerstrand does not necessarily refer to a continuous single strandednucleic acid and may comprise a discontinuity, preferably at a site thatis cleaved by Dicer. A passenger strand is a sense strand.

By “target nucleic acid” is meant a nucleic acid sequence whoseexpression, level or activity is to be modulated. The target nucleicacid can be DNA or RNA. For agents that target Glycolate Oxidase, incertain embodiments, the target nucleic acid is Glycolate Oxidase (HAO1)RNA, e.g., in certain embodiments, Glycolate Oxidase (HAO1) mRNA.Glycolate Oxidase RNA target sites can also interchangeably bereferenced by corresponding cDNA sequences. Levels of Glycolate Oxidasemay also be targeted via targeting of upstream effectors of GlycolateOxidase, or the effects of modulated or misregulated Glycolate Oxidasemay also be modulated by targeting of molecules downstream of GlycolateOxidase in the Glycolate Oxidase signalling pathway.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, a dsRNA moleculeof the invention comprises 19 to 30 (e.g., 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 or more) nucleotides that are complementary to oneor more target nucleic acid molecules or a portion thereof.

As used herein, a dsNA, e.g., DsiRNA or siRNA, having a sequence“sufficiently complementary” to a target RNA or cDNA sequence (e.g.,glycolate oxidase (HAO1) mRNA) means that the dsNA has a sequencesufficient to trigger the destruction of the target RNA (where a cDNAsequence is recited, the RNA sequence corresponding to the recited cDNAsequence) by the RNAi machinery (e.g., the RISC complex) or process. Forexample, a dsNA that is “sufficiently complementary” to a target RNA orcDNA sequence to trigger the destruction of the target RNA by the RNAimachinery or process can be identified as a dsNA that causes adetectable reduction in the level of the target RNA in an appropriateassay of dsNA activity (e.g., an in vitro assay as described in Example2 below), or, in further examples, a dsNA that is sufficientlycomplementary to a target RNA or cDNA sequence to trigger thedestruction of the target RNA by the RNAi machinery or process can beidentified as a dsNA that produces at least a 5%, at least a 10%, atleast a 15%, at least a 20%, at least a 25%, at least a 30%, at least a35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, atleast a 60%, at least a 65%, at least a 70%, at least a 75%, at least a80%, at least a 85%, at least a 90%, at least a 95%, at least a 98% orat least a 99% reduction in the level of the target RNA in anappropriate assay of dsNA activity. In additional examples, a dsNA thatis sufficiently complementary to a target RNA or cDNA sequence totrigger the destruction of the target RNA by the RNAi machinery orprocess can be identified based upon assessment of the duration of acertain level of inhibitory activity with respect to the target RNA orprotein levels in a cell or organism. For example, a dsNA that issufficiently complementary to a target RNA or cDNA sequence to triggerthe destruction of the target RNA by the RNAi machinery or process canbe identified as a dsNA capable of reducing target mRNA levels by atleast 20% at least 48 hours post-administration of said dsNA to a cellor organism. Preferably, a dsNA that is sufficiently complementary to atarget RNA or cDNA sequence to trigger the destruction of the target RNAby the RNAi machinery or process is identified as a dsNA capable ofreducing target mRNA levels by at least 40% at least 72 hourspost-administration of said dsNA to a cell or organism, by at least 40%at least four, five or seven days post-administration of said dsNA to acell or organism, by at least 50% at least 48 hours post-administrationof said dsNA to a cell or organism, by at least 50% at least 72 hourspost-administration of said dsNA to a cell or organism, by at least 50%at least four, five or seven days post-administration of said dsNA to acell or organism, by at least 80% at least 48 hours post-administrationof said dsNA to a cell or organism, by at least 80% at least 72 hourspost-administration of said dsNA to a cell or organism, or by at least80% at least four, five or seven days post-administration of said dsNAto a cell or organism.

In certain embodiments, a nucleic acid of the invention (e.g., a DsiRNAor siRNA) possesses a sequence “sufficiently complementary to hybridize”to a target RNA or cDNA sequence, thereby achieving an inhibitory effectupon the target RNA. Hybridization, and conditions available fordetermining whether one nucleic acid is sufficiently complementary toanother nucleic acid to allow the two sequences to hybridize, isdescribed in greater detail below.

As will be clear to one of ordinary skill in the art, “sufficientlycomplementary” (contrasted with, e.g., “100% complementary”) allows forone or more mismatches to exist between a dsNA of the invention and thetarget RNA or cDNA sequence (e.g., glycolate oxidase (HAO1) mRNA),provided that the dsNA possesses complementarity sufficient to triggerthe destruction of the target RNA by the RNAi machinery (e.g., the RISCcomplex) or process. In certain embodiments, a “sufficientlycomplementary” dsNA of the invention can harbor one, two, three or evenfour or more mismatches between the dsNA sequence and the target RNA orcDNA sequence (e.g., in certain such embodiments, the antisense strandof the dsRNA harbors one, two, three, four, five or even six or moremismatches when aligned with the target RNA or cDNA sequence for maximumcomplementarity). Additional consideration of the preferred location ofsuch mismatches within certain dsRNAs of the instant invention isconsidered in greater detail below.

In one embodiment, dsRNA molecules of the invention that down regulateor reduce Glycolate Oxidase gene expression are used for treating,preventing or reducing Glycolate Oxidase-related diseases or disorders(e.g., PH1) in a subject or organism.

In one embodiment of the present invention, each sequence of a DsiRNAmolecule of the invention is independently 25 to 35 nucleotides inlength, in specific embodiments 25, 26, 27, 28, 29, 30, 31, 32, 33, 34or 35 nucleotides in length. In another embodiment, the DsiRNA duplexesof the invention independently comprise 25 to 30 base pairs (e.g., 25,26, 27, 28, 29, or 30). In another embodiment, one or more strands ofthe DsiRNA molecule of the invention independently comprises 19 to 35nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34 or 35) that are complementary to a target (Glycolate Oxidase)nucleic acid molecule. In certain embodiments, a DsiRNA molecule of theinvention possesses a length of duplexed nucleotides between 25 and 66nucleotides, optionally between 25 and 49 nucleotides in length (e.g.,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48 or 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66 nucleotides in length; optionally, all suchnucleotides base pair with cognate nucleotides of the opposite strand).In related embodiments, a dsNA of the invention possesses strand lengthsthat are, independently, between 19 and 66 nucleotides in length,optionally between 25 and 53 nucleotides in length, e.g., 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48 or 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66 nucleotides in length. In certainembodiments, one strand length is 19-35 nucleotides in length, while theother strand length is 30-66 nucleotides in length and at least onestrand has a 5′ overhang of at least 5 nucleotides in length relative tothe other strand. In certain related embodiments, the 3′ end of thefirst strand and the 5′ end of the second strand form a structure thatis a blunt end or a 1-6 nucleotide 3′ overhang, while the 5′ end of thefirst strand forms a 5-35 nucleotide overhang with respect to the 3′ endof the second strand. Optionally, between one and all nucleotides of the5-35 nucleotide overhang are modified nucleotides (optionally,deoxyribonucleotides and/or modified ribonucleotides).

In some embodiments, a dsNA of the invention has a first or secondstrand that has at least 8 contiguous ribonucleotides. In certainembodiments, a dsNA of the invention has 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 or more (e.g., 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 26, or more, up to the full length of the strand)ribonucleotides, optionally including modified ribonucleotides(2′-O-methyl ribonucleotides, phosphorothioate linkages, etc.). Incertain embodiments, the ribonucleotides or modified ribonucleotides arecontiguous.

In certain embodiments of the invention, tetraloop- and modifiednucleotide-containing dsNAs are contemplated as described, e.g., in US2011/0288147. In certain such embodiments, a dsNA of the inventionpossesses a first strand and a second strand, where the first strand andthe second strand form a duplex region of 19-25 nucleotides in length,wherein the first strand comprises a 3′ region that extends beyond thefirst strand-second strand duplex region and comprises a tetraloop, andthe dsNA comprises a discontinuity between the 3′ terminus of the firststrand and the 5′ terminus of the second strand. Optionally, thediscontinuity is positioned at a projected dicer cleavage site of thetetraloop-containing dsNA. It is contemplated that, as for any of theother dupexed oligonucleotides of the invention, tetraloop-containingduplexes of the invention can possess any range of modificationsdisclosed herein or otherwise known in the art, including, e.g.,2′-O-methyl, 2′-fluoro, inverter basic, GalNAc moieties, etc.

In certain embodiments, a dsNA comprising a first strand and a secondstrand, each strand, independently, having a 5′ terminus and a 3′terminus, and having, independently, respective strand lengths of 25-53nucleotides in length, is sufficiently highly modified (e.g., at least10% or more, at least 20% or more, at least 30% or more, at least 40% ormore, at least 50% or more, at least 60% or more, at least 70% or more,at least 80% or more, at least 90% or more, at least 95% or moreresidues of one and/or both strands are modified such that dicercleavage of the dsNA is prevented (optionally, modified residues occurat and/or flanking one or all predicted dicer cleavage sites of thedsNA). Such non-dicer-cleaved dsNAs retain glycolate oxidase (HAO1)inhibition activity and are optionally cleaved by non-dicer nucleases toyield, e.g., 15-30, or in particular embodiments, 19-23 nucleotidestrand length dsNAs capable of inhibiting glycolate oxidase (HAO1) in amammalian cell. In certain related embodiments, dsNAs possessingsufficiently extensive modification to block dicer cleavage of suchdsNAs optionally possess regions of unmodified nucleotide residues(e.g., one or two or more consecutive nucleotides, forming a “gap” or“window” in a modification pattern) that allow for and/or promotecleavage of such dsNAs by non-Dicer nucleases. In other embodiments,Dicer-cleaved dsNAs of the invention can include extensive modificationpatterns that possess such “windows” or “gaps” in modification such thatDicer cleavage preferentially occurs at such sites (as compared toheavily modified regions within such dsNAs).

In certain embodiments of the present invention, an oligonucleotide isprovided (optionally, as a free antisense oligonucleotide or as anoligonucleotide of a double-stranded or other multiple-strandedstructure) that includes a sequence complementary to the HAO1 target asdescribed elsewhere herein and that is 15 to 80 nucleotides in length,e.g., in specific embodiments 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79 or 80 nucleotides in length.

In certain additional embodiments of the present invention, eacholigonucleotide of a DsiRNA molecule of the invention is independently25 to 53 nucleotides in length, in specific embodiments 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52 or 53 nucleotides in length. For DsiRNAspossessing a strand that exceeds 30 nucleotides in length, availablestructures include those where only one strand exceeds 30 nucleotides inlength (see, e.g., U.S. Pat. No. 8,349,809), or those where both strandsexceed 30 nucleotides in length (see, e.g., WO 2010/080129). Stabilizingmodifications (e.g., 2′-O-Methyl, phosphorothioate,deoxyribonucleotides, including dNTP base pairs, 2′-F, etc.) can beincorporated within any double stranded nucleic acid of the invention,and can be used in particular, and optionally in abundance, especiallywithin DsiRNAs possessing one or both strands exceeding 30 nucleotidesin length. While the guide strand of a double stranded nucleic acid ofthe invention must possess a sequence of, e.g., 15, 16, 17, 18 or 19nucleotides that are complementary to a target RNA (e.g., mRNA),additional sequence(s) of the guide strand need not be complementary tothe target RNA. The end structures of double stranded nucleic acidspossessing at least one strand length in excess of 30 nucleotides canalso be varied while continuing to yield functional dsNAs—e.g. the 5′end of the guide strand and the 3′ end of the passenger strand may forma 5′-overhang, a blunt end or a 3′ overhang (for certain dsNAs, e.g.,“single strand extended” dsNAs, the length of such a 5′ or 3′ overhangcan be 1-4, 1-5, 1-6, 1-10, 1-15, 1-20 or even 1-25 or morenucleotides); similarly, the 3′ end of the guide strand and the 5′ endof the passenger strand may form a 5′-overhang, a blunt end or a 3′overhang (for certain dsNAs, e.g., “single strand extended” dsNAs, thelength of such a 5′ or 3′ overhang can be 1-4, 1-5, 1-6, 1-10, 1-15,1-20 or even 1-25 or more nucleotides). In certain embodiments, the 5′end of the passenger strand includes a 5′-overhang relative to the 3′end of the guide strand, such that a one to fifteen or more nucleotidesingle strand extension exists. Optionally, such single strandextensions of the dsNAs of the invention (whether present on thepassenger or the guide strand) can be modified, e.g., withphosphorothioate (PS), 2′-F, 2′-O-methyl and/or other forms ofmodification contemplated herein or known in the art, includingconjugation to, e.g., GalNAc moieties, inverted abasic residues, etc. Insome embodiments, the guide strand of a single-strand extended duplex ofthe invention is between 35 and 50 nucleotides in length, while theduplex presents a single-strand extension that is seven to twentynucleotides in length. In certain embodiments, the guide strand of aduplex is between 37 and 42 nucleotides in length and the duplexpossesses a 5′ single-stranded overhang of the passenger strand that isapproximately five to fifteen single-stranded nucleotides in length(optionally, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length).In related embodiments, the passenger strand of the duplex is about 25to about 30 nucleotides in length. In certain embodiments, the extendedregion of the duplex can optionally be extensively modified, e.g., withone or more of 2′-O-methyl, 2′-Fluoro, GalNAc moieties, phosphorothioateinternucleotide linkages, inverted abasic residues, etc. In certainembodiments, the length of the passenger strand is 31-49 nucleotideswhile the length of the guide strand is 31-53 nucleotides, optionallywhile the 5′ end of the guide strand forms a blunt end (optionally, abase-paired blunt end) with the 3′ end of the passenger strand,optionally, with the 3′ end of the guide strand and the 5′ end of thepassenger strand forming a 3′ overhang of 1-4 nucleotides in length.Exemplary “extended” Dicer substrate structures are set forth, e.g., inUS 2010/0173974 U.S. Pat. No. 8,513,207 and U.S. Pat. No. 8,349,809,both of which are incorporated herein by reference. In certainembodiments, one or more strands of the dsNA molecule of the inventionindependently comprises 19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementaryto a target (Glycolate Oxidase) nucleic acid molecule. In certainembodiments, a DsiRNA molecule of the invention possesses a length ofduplexed nucleotides between 25 and 49 nucleotides in length (e.g., 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48 or 49 nucleotides in length; optionally, all suchnucleotides base pair with cognate nucleotides of the opposite strand).

In a further embodiment of the present invention, each oligonucleotideof a DsiRNA molecule of the invention is independently 19 to 66nucleotides in length, in specific embodiments 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65 or 66 nucleotides in length. For dsNAs possessing astrand that exceeds 30 nucleotides in length, available structuresinclude those where only one strand exceeds 30 nucleotides in length(see, e.g., U.S. Pat. No. 8,349,809, where an exemplary double strandednucleic acid possesses a first oligonucleotide strand having a 5′terminus and a 3′ terminus and a second oligonucleotide strand having a5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′terminal nucleotide and each of the 3′ termini has a 3′ terminalnucleotide, where the first strand (or the second strand) is 25-30nucleotide residues in length, where starting from the 5′ terminalnucleotide (position 1) positions 1 to 23 of the first strand (or thesecond strand) include at least 8 ribonucleotides; the second strand (orthe first strand) is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, includes at least 8 ribonucleotides inthe positions paired with positions 1-23 of the first strand to form aduplex; where at least the 3′ terminal nucleotide of the second strand(or the first strand) is unpaired with the first strand (or the secondstrand), and up to 6 consecutive 3′ terminal nucleotides are unpairedwith the first strand (or the second strand), thereby forming a 3′single stranded overhang of 1-6 nucleotides; where the 5′ terminus ofthe second strand (or the first strand) includes from 10-30 consecutivenucleotides which are unpaired with the first strand (or the secondstrand), thereby forming a 10-30 nucleotide single stranded 5′ overhang;where at least the first strand (or the second strand) 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of the secondstrand (or first strand) when the first and second strands are alignedfor maximum complementarity, thereby forming a substantially duplexedregion between the first and second strands; and the second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell), or those where both strands exceed 30 nucleotides inlength (see, e.g., U.S. Pat. No. 8,513,207, where an exemplary doublestranded nucleic acid (dsNA) possesses a first oligonucleotide strandhaving a 5′ terminus and a 3′ terminus and a second oligonucleotidestrand having a 5′ terminus and a 3′ terminus, where the first strand is31 to 49 nucleotide residues in length, where starting from the firstnucleotide (position 1) at the 5′ terminus of the first strand,positions 1 to 23 of the first strand are ribonucleotides; the secondstrand is 31 to 53 nucleotide residues in length and includes 23consecutive ribonucleotides that base pair with the ribonucleotides ofpositions 1 to 23 of the first strand to form a duplex; the 5′ terminusof the first strand and the 3′ terminus of said second strand form ablunt end or a 1-4 nucleotide 3′ overhang; the 3′ terminus of the firststrand and the 5′ terminus of said second strand form a duplexed bluntend, a 5′ overhang or a 3′ overhang; optionally, at least one ofpositions 24 to the 3′ terminal nucleotide residue of the first strandis a deoxyribonucleotide, optionally, that base pairs with adeoxyribonucleotide of said second strand; and the second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell).

In certain embodiments, an active dsNA of the invention can possess a 5′overhang of the first strand (optionally, the passenger strand) withrespect to the second strand (optionally, the guide strand) of 2-50nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or morein length. In related embodiments, the duplex region formed by the firstand second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more base pairs in length. The 5′ overhang “extended” regionof the first strand is optionally modified at one or more residues(optionally, at alternating residues, all residues, or any otherselection of residues).

In certain embodiments, an active dsNA of the invention can possess a 3′overhang of the first strand (optionally, the passenger strand) withrespect to the second strand (optionally, the guide strand) of 2-50nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or morein length. In related embodiments, the duplex region formed by the firstand second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more base pairs in length. The 3′ overhang “extended” regionof the first strand is optionally modified at one or more residues(optionally, at alternating residues, all residues, or any otherselection of residues).

In additional embodiments, an active dsNA of the invention can possess a5′ overhang of the second strand (optionally, the guide strand) withrespect to the first strand (optionally, the passenger strand) of 2-50nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or morein length. In related embodiments, the duplex region formed by the firstand second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more base pairs in length. The 5′ overhang “extended” regionof the second strand is optionally modified at one or more residues(optionally, at alternating residues, all residues, or any otherselection of residues).

In further embodiments, an active dsNA of the invention can possess a 3′overhang of the second strand (optionally, the guide strand) withrespect to the first strand (optionally, the passenger strand) of 2-50nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or morein length. In related embodiments, the duplex region formed by the firstand second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more base pairs in length. The 3′ overhang “extended” regionof the second strand is optionally modified at one or more residues(optionally, at alternating residues, all residues, or any otherselection of residues).

In another embodiment of the present invention, each sequence of aDsiRNA molecule of the invention is independently 25 to 35 nucleotidesin length, in specific embodiments 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 nucleotides in length. In another embodiment, the DsiRNAduplexes of the invention independently comprise 25 to 30 base pairs(e.g., 25, 26, 27, 28, 29, or 30). In another embodiment, one or morestrands of the DsiRNA molecule of the invention independently comprises19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34 or 35) that are complementary to a target (HAO1)nucleic acid molecule. In certain embodiments, a DsiRNA molecule of theinvention possesses a length of duplexed nucleotides between 25 and 34nucleotides in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34nucleotides in length; optionally, all such nucleotides base pair withcognate nucleotides of the opposite strand). (Exemplary DsiRNA moleculesof the invention are shown in FIG. 1, and below.)

In certain embodiments, at least 10%, at least 20%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or more of the nucleotide residuesof a nucleic acid of the instant invention are modified residues. For adsNA of the invention, at least 10%, at least 20%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or more of the nucleotide residuesof the first strand are modified residues. Additionally and/oralternatively for a dsNA of the invention, at least 10%, at least 20%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or more of thenucleotide residues of the second strand are modified residues. For thedsNAs of the invention, modifications of both duplex (double-stranded)regions and overhang (single-stranded) regions are contemplated. Thus,in certain embodiments, at least 10%, at least 20%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or more (e.g., all) duplexnucleotide residues are modified residues. Additionally and/oralternatively, at least 10%, at least 20%, at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or more (e.g., all) overhang nucleotide residuesof one or both strands are modified residues. Optionally, themodifications of the dsNAs of the invention do not include an invertedabasic (e.g., inverted deoxy abasic) or inverted dT end-protectinggroup. Alternatively, a dsNA of the invention includes a terminal capmoiety (e.g., an inverted deoxy abasic and/or inverted dT end-protectinggroup). Optionally, such a terminal cap moiety is located at the 5′ end,at the 3′ end, or at both the 5′ end and the 3′ end of the first strand,of the second strand, or of both first and second strands.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell. Within certainaspects, the term “cell” refers specifically to mammalian cells, such ashuman cells, that contain one or more isolated dsRNA molecules of thepresent disclosure. In particular aspects, a cell processes dsRNAs ordsRNA-containing molecules resulting in RNA intereference of targetnucleic acids, and contains proteins and protein complexes required forRNAi, e.g., Dicer and RISC.

In certain embodiments, dsRNAs of the invention are Dicer substratesiRNAs (“DsiRNAs”). DsiRNAs can possess certain advantages as comparedto inhibitory nucleic acids that are not dicer substrates(“non-DsiRNAs”). Such advantages include, but are not limited to,enhanced duration of effect of a DsiRNA relative to a non-DsiRNA, aswell as enhanced inhibitory activity of a DsiRNA as compared to anon-DsiRNA (e.g., a 19-23 mer siRNA) when each inhibitory nucleic acidis suitably formulated and assessed for inhibitory activity in amammalian cell at the same concentration (in this latter scenario, theDsiRNA would be identified as more potent than the non-DsiRNA).Detection of the enhanced potency of a DsiRNA relative to a non-DsiRNAis often most readily achieved at a formulated concentration (e.g.,transfection concentration of the dsRNA) that results in the DsiRNAeliciting approximately 30-70% knockdown activity upon a target RNA(e.g., a mRNA). For active DsiRNAs, such levels of knockdown activityare most often achieved at in vitro mammalian cell DsiRNA transfectionconcentrations of 1 nM or less of as suitably formulated, and in certaininstances are observed at DsiRNA transfection concentrations of 200 pMor less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5pM or less, or even 1 pM or less. Indeed, due to the variability amongDsiRNAs of the precise concentration at which 30-70% knockdown of atarget RNA is observed, construction of an IC₅₀ curve via assessment ofthe inhibitory activity of DsiRNAs and non-DsiRNAs across a range ofeffective concentrations is a preferred method for detecting theenhanced potency of a DsiRNA relative to a non-DsiRNA inhibitory agent.

In certain embodiments, a DsiRNA (in a state as initially formed, priorto dicer cleavage) is more potent at reducing Glycolate Oxidase targetgene expression in a mammalian cell than a 19, 20, 21, 22 or 23 basepair sequence that is contained within it. In certain such embodiments,a DsiRNA prior to dicer cleavage is more potent than a 19-21 mercontained within it. Optionally, a DsiRNA prior to dicer cleavage ismore potent than a 19 base pair duplex contained within it that issynthesized with symmetric dTdT overhangs (thereby forming a siRNApossessing 21 nucleotide strand lengths having dTdT overhangs). Incertain embodiments, the DsiRNA is more potent than a 19-23 mer siRNA(e.g., a 19 base pair duplex with dTdT overhangs) that targets at least19 nucleotides of the 21 nucleotide target sequence that is recited fora DsiRNA of the invention (without wishing to be bound by theory, theidentity of a such a target site for a DsiRNA is identified viaidentification of the Ago2 cleavage site for the DsiRNA; once the Ago2cleavage site of a DsiRNA is determined for a DsiRNA, identification ofthe Ago2 cleavage site for any other inhibitory dsRNA can be performedand these Ago2 cleavage sites can be aligned, thereby determining thealignment of projected target nucleotide sequences for multiple dsRNAs).In certain related embodiments, the DsiRNA is more potent than a 19-23mer siRNA that targets at least 20 nucleotides of the 21 nucleotidetarget sequence that is recited for a DsiRNA of the invention.Optionally, the DsiRNA is more potent than a 19-23 mer siRNA thattargets the same 21 nucleotide target sequence that is recited for aDsiRNA of the invention. In certain embodiments, the DsiRNA is morepotent than any 21 mer siRNA that targets the same 21 nucleotide targetsequence that is recited for a DsiRNA of the invention. Optionally, theDsiRNA is more potent than any 21 or 22 mer siRNA that targets the same21 nucleotide target sequence that is recited for a DsiRNA of theinvention. In certain embodiments, the DsiRNA is more potent than any21, 22 or 23 mer siRNA that targets the same 21 nucleotide targetsequence that is recited for a DsiRNA of the invention. As noted above,such potency assessments are most effectively performed upon dsRNAs thatare suitably formulated (e.g., formulated with an appropriatetransfection reagent) at a concentration of 1 nM or less. Optionally, anIC₅₀ assessment is performed to evaluate activity across a range ofeffective inhibitory concentrations, thereby allowing for robustcomparison of the relative potencies of dsRNAs so assayed.

The dsRNA molecules of the invention are added directly, or can becomplexed with lipids (e.g., cationic lipids), packaged withinliposomes, or otherwise delivered to target cells or tissues. Thenucleic acid or nucleic acid complexes can be locally administered torelevant tissues ex vivo, or in vivo through direct dermal application,transdermal application, or injection, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in FIG. 1, andthe below exemplary structures. Examples of such nucleic acid moleculesconsist essentially of sequences defined in these figures and exemplarystructures. Furthermore, where such agents are modified in accordancewith the below description of modification patterning of DsiRNA agents,chemically modified forms of constructs described in FIG. 1, and thebelow exemplary structures can be used in all uses described for theDsiRNA agents of FIG. 1, and the below exemplary structures.

In another aspect, the invention provides mammalian cells containing oneor more dsRNA molecules of this invention. The one or more dsRNAmolecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one, and preferably atleast 4, 8 and 12 ribonucleotide residues. The at least 4, 8 or 12 RNAresidues may be contiguous. By “ribonucleotide” is meant a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.The terms include double-stranded RNA, single-stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the dsRNAor internally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the dsRNA agents of the invention can be administered.A subject can be a mammal or mammalian cells, including a human or humancells.

The phrase “pharmaceutically acceptable carrier” refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA compositions may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (seeKreuter, 1991). The polymeric materials which are formed from monomericand/or oligomeric precursors in the polymerization/nanoparticlegeneration step, are per se known from the prior art, as are themolecular weights and molecular weight distribution of the polymericmaterial which a person skilled in the field of manufacturingnanoparticles may suitably select in accordance with the usual skill.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isa control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent (e.g., DsiRNA) of theinvention into a cell or organism. In another embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined in a cell or organism, e.g., acontrol or normal cell or organism, exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a dsRNA agent or avector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease or disorder, or symptoms of the disease or disorder. The term“treatment” or “treating” is also used herein in the context ofadministering agents prophylactically. The term “effective dose” or“effective dosage” is defined as an amount sufficient to achieve or atleast partially achieve the desired effect. The term “therapeuticallyeffective dose” is defined as an amount sufficient to cure or at leastpartially arrest the disease and its complications in a patient alreadysuffering from the disease. The term “patient” includes human and othermammalian subjects that receive either prophylactic or therapeutictreatment.

Structures of Anti-Glycolate Oxidase DsiRNA Agents

In certain embodiments, the anti-Glycolate Oxidase DsiRNA agents of theinvention can have the following structures:

In one such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “Y” is an overhang domain comprised of 1-4 RNAmonomers that are optionally 2′-O-methyl RNA monomers. In a relatedembodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “Y” is an overhang domain comprised of 1-4 RNAmonomers that are optionally 2′-O-methyl RNA monomers, and “D”=DNA. Inone embodiment, the top strand is the sense strand, and the bottomstrand is the antisense strand. Alternatively, the bottom strand is thesense strand and the top strand is the antisense strand.

DsiRNAs of the invention can carry a broad range of modificationpatterns (e.g., 2′-O-methyl RNA patterns, e.g., within extended DsiRNAagents). Certain modification patterns of the second strand of DsiRNAsof the invention are presented below.

In one embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. In arelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. In arelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. In arelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. In arelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. In a further relatedembodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M7” or “M7”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. In arelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. The top strand is the sense strand, and the bottomstrand is the antisense strand. In another related embodiment, theDsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M6” or “M6”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In anotherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M5” or “M5”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M4” or “M4”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M8” or “M8”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M3” or “M3”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M2” or “M2”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M1” or “M1”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M9” or “M9”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M10” or “M10”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M11” or “M11”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M12” or “M12”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M13” or “M13”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M21” or “M21”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M14” or “M14”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M15” or “M15”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M16” or “M16”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M17” or “M17”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M18” or “M18”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M19” or “M19”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M20” or “M20”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M22” or “M22”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M24” or “M24”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M25” or “M25”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M26” or “M26”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M27” or “M27”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M28” or “M28”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M29” or “M29”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M30” or “M30”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M31” or “M31”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M32” or “M32”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M34” or “M34”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M35” or “M35”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M37” or “M37”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M38” or “M38”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M40” or “M40”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M41” or “M41”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M36” or “M36”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M42” or “M42”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M43” or “M43”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M44” or “M44”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M45” or “M45”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M46” or “M46”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M47” or “M47”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M48” or “M48”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M52” or “M52”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M54” or “M54”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M55” or “M55”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M56” or “M56”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M57” or “M57”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M58” or “M58”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M59” or “M59”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M60” or “M60”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M61” or “M61”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M62” or “M62”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M63” or “M63”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M64” or “M64”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M65” or “M65”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M66” or “M66”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M67” or “M67”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M68” or “M68”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M69” or “M69”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M70” or “M70”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M71” or “M71”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M72” or “M72”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers and underlined residues are 2′-O-methyl RNA monomers. The topstrand is the sense strand, and the bottom strand is the antisensestrand. In one related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M73” or “M73”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. In a further relatedembodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M7*” or “M7*”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M6*” or “M6*”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In anotherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M5*” or “M5*”modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M4*” or “M4*”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M8*” or “M8*”modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M2*” or “M2*”modification pattern.

In other embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M10*” or“M10*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M11*” or“M11*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M13*” or“M13*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M14*” or“M14*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M15*” or“M15*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M16*” or“M16*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M17*” or“M17*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M18*” or“M18*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M19*” or“M19*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA.The top strand is the sense strand, and the bottom strand is theantisense strand. In another related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 33′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M20*” or“M20*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M22*” or“M22*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M24*” or“M24*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M25*” or“M25*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M26*” or“M26*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M27*” or“M27*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M28*” or“M28*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M29*” or“M29*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M34*” or“M34*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M35*” or“M35*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M37*” or“M37*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M38*” or“M38*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M40*” or“M40*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M41*” or“M41*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M36*” or“M36*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M42*” or“M42*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M43*” or“M43*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M44*” or“M44*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M46*” or“M46*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M47*” or“M47*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M48*” or“M48*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M52*” or“M52*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M54*” or“M54*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M55*” or“M55*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M56*” or“M56*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M57*” or“M57*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M58*” or“M58*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M59*” or“M59*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M60*” or“M60*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M61*” or“M61*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M62*” or“M62*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M63*” or“M63*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M64*” or“M64*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M65*” or“M65*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M66*” or“M66*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M67*” or“M67*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M68*” or“M68*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M69*” or“M69*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M70*” or“M70*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M71*” or“M71*” modification pattern.

In further embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M72*” or“M72*” modification pattern.

In additional embodiments, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”=RNA and “X”, 2′-O-methyl RNA. The top strand is the sensestrand, and the bottom strand is the antisense strand. In a furtherrelated embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “X”, 2′-O-methyl RNA, and “D”=DNA. The top strand isthe sense strand, and the bottom strand is the antisense strand. Thismodification pattern is also referred to herein as the “AS-M73*” or“M73*” modification pattern.

Additional exemplary antisense strand modifications include thefollowing:

3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M74”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M75”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M76”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M77”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M78”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M79”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M80”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M81”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M82”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M83”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M84”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M85”3′-FFFXXXXXXXXXXXXXXXXXXXXFFFF-5′ “AS-M88”3′-XXXXFXXXFXFXXXXXXXXXXXXXXXX-5′ “AS-M89”3′-FFFXFXFXFXFXFXFXFXXXXXXFFFF-5′ “AS-M90”3′-FFFXXXXXXXXXXXXXXXXXXXXXXFF-5′ “AS-M91”3′-XXXXXXFXXXXXFXFXXXXXXXXXXXX-5′ “AS-M92”3′-FFFXFXXXXXXXXXXXXXFXXXXXXXX-5′ “AS-M93”3′-FFFXFXFXXXXXFXFXFXFXXXXFFFF-5′ “AS-M94”3′-FFFXFXFXFXFXFXFXFXXXXXXFFFF-5′ “AS-M95”3′-FFFXFXFXFXFXFXFXFXXXXXXFFFpF-5′ “AS-M96”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M210”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M74*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M75*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M76*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M77*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M78*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M79*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M80*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M82*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M83*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M84*”3′-XFFXXXXXXXXXXXXXXXXXXXXFFFF-5′ “AS-M88*”3′-XXXXFXXXFXFXXXXXXXXXXXXXXXX-5′ “AS-M89*”3′-XFFXFXFXFXFXFXFXFXXXXXXFFFF-5′ “AS-M90*”3′-XFFXXXXXXXXXXXXXXXXXXXXXXFF-5′ “AS-M91*”3′-XXXXXXFXXXXXFXFXXXXXXXXXXXX-5′ “AS-M92*”3′-XFFXFXXXXXXXXXXXXXFXXXXXXXX-5′ “AS-M93*”3′-XFFXFXFXXXXXFXFXFXFXXXXFFFF-5′ “AS-M94*”3′-XFFXFXFXFXFXFXFXFXXXXXXFFFF-5′ “AS-M95*”3′-XFFXFXFXFXFXFXFXFXXXXXXFFFpF-5′ “AS-M96*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M210*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M101”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M104”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M104*”3′-XpFpXFXFXFXFXFXFXFXFXFXFFXXXpX-5′ “AS-M105”3′-XpFpXFXFXFXFXFXFXFXFXFXFFXXXpX-5′ “AS-M105*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M106”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M106*”3′-XXpXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M107”3′-XXpXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M107*”3′-ba-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M108” (ba =inverted abasic for F7 stabilization at 3′ end)3′-ba-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M108*” (ba =inverted abasic for F7 stabilization at 3′ end)3′-XDXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M109”3′-XpXXFXFXFXXXXXXXXXXXXXXXXXXpX-5′ “AS-M110”3′-XpXXFXFXFXXXXXXXXXXXXXXXXXXpX-5′ “AS-M110*”3′-XpXXFXFXFXXXXXFXFXFXFXXXXXXpX-5′ “AS-M111”3′-XpXXFXFXFFXXXXXXXXXXFXXXXXXpX-5′ “AS-M112”3′-XpXXFXFXFFXXFXXXXXXXFXXXXXXpX-5′ “AS-M113”3′-XpXXFXFXFFFFFXXXXXXXFXXXXXXpX-5′ “AS-M114”3′-XpXXFXFXFFFFFXXXXXXXFXXFXXXpX-5′ “AS-M115”3′-XpXXFXFXFFFFFXXXXXXXFFFFXXXpX-5′ “AS-M116”3′-XpFXFXFXFXFXFXFXFXFXFXFXFXFpX-5′ “AS-M117”3′-XpXXFXFXFXXXXXFXFXFXFXXXXXXpX-5′ “AS-M111*”3′-XpXXFXFXFFXXXXXXXXXXFXXXXXXpX-5′ “AS-M112*”3′-XpXXFXFXFFXXFXXXXXXXFXXXXXXpX-5′ “AS-M113*”3′-XpXXFXFXFFFFFXXXXXXXFXXXXXXpX-5′ “AS-M114*”3′-XpXXFXFXFFFFFXXXXXXXFXXFXXXpX-5′ “AS-M115*”3′-XpXXFXFXFFFFFXXXXXXXFFFFXXXpX-5′ “AS-M116*”3′-XpFXFXFXFXFXFXFXFXFXFXFXFXFpX-5′ “AS-M117*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M120”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M121”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M122”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M123”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M124”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M125”3′-XpFpXFXFXFXXXXXFXFXFXFXXXFXFpX-5′ “AS-M126”3′-XpFpXFXFXFXXXXXFXFXFXFXXXFXFpX-5′ “AS-M127”3′-XpFpXFXFXFXFXFXFXFXFXFXFFXXXpX-5′ “AS-M128”3′-XpFpXFXFXFXFXFXFXFXFXFXFFFFFpF-5′ “AS-M129”3′-XpFpXFXFXFXFXFXFXFXFXFXXXFXFpX-5′ “AS-M130”3′-XpFpXFXFXFXFXFXFXFXFXFXFXFXFpX-5′ “AS-M131”3′-XpFpXFXFXFXFXFXFXFXFXFXFXXXXpX-5′ “AS-M132”3′-XpFpXFXFXFXFXFXFXFXFXFXFXXXXpX-5′ “AS-M133”3′-XpFpXFXFXFXFXFXFXFXFXFXFXFFFpF-5′ “AS-M134”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M135”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M136”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M137”3′-XXXFXFXFXXXXXFXFXFXFXXXXXXX-5′ “AS-M138”3′-XXXFXFXFFXXXXXXXXXXFXXXXXXX-5′ “AS-M139”3′-XpXXFXFXFXXXXXFXFXFFFXXXXXXpX-5′ “AS-M140”3′-XpXXFXFXFFXXXFFXFXFFFXXXXXXpX-5′ “AS-M141”3′-XpXXFXFXFXFXFXFXFXFFFXXXXXXpX-5′ “AS-M142”3′-XpXXFXFXFFFFFFFXFXFFFXXXXXXpX-5′ “AS-M143”3′-XpXXFXFXFXFXFXFXFXFpXpFpXXXXXXpX-5′ “AS-M144”3′-XXXXXXXXXXXXXXXXFXXXXXXXXXX-5′ “AS-M145”3′-XXXXXXXXXXXXXXFXXXXXXXXXXXX-5′ “AS-M146”3′-XXXXXXFXXXXXXXXXXXXXXXXXXXX-5′ “AS-M147”3′-XXXXXXXXXXFXXXXXXXXXXXXXXXX-5′ “AS-M148”3′-XXXXXXXXXXXXFXXXXXXXXXXXXXX-5′ “AS-M149”3′-XXXXXXXXXXFXFXXXXXXXXXXXXXX-5′ “AS-M150”3′-XXXXXXXXXXXXFXXXFXXXXXXXXXX-5′ “AS-M151”3′-XXXXXXXXXXFXXXFXXXXXXXXXXXX-5′ “AS-M152”3′-XXXXXXXXFXXXXXXXXXXXXXXXXXX-5′ “AS-M153”3′-XXXXXXXXFXXXXXXXXXXXXXXXXXX-5′ “AS-M154”3′-XXXXXXFXXXXXXXXXXXXXXXXXXXX-5′ “AS-M155”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M156”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M157”3′-XpXXFXFFFFXXXFXXFXXFFXXXXXXpX-5′ “AS-M158”3′-XpXXFXFFFFFFFFXXFXXFFXXXXXXpX-5′ “AS-M159”3′-XpXXFXXXFXXXXXFXFXXFFXXXXXXpX-5′ “AS-M160”3′-XpXXFXXXFXFXFXFXFXXFFXXXXXXpX-5′ “AS-M161”3′-XXXXXXXXFXFXFXXXXXXXXXXXXXX-5′ “AS-M162”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M163”3′-XXXXXXXXDXXXXXXXXXXXXXXXXXX-5′ “AS-M164”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M120*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M121*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M122*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M123*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M124*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M125*”3′-XpFpXFXFXFXXXXXFXFXFXFXXXFXFpX-5′ “AS-M126*”3′-XpFpXFXFXFXXXXXFXFXFXFXXXFXFpX-5′ “AS-M127*”3′-XpFpXFXFXFXFXFXFXFXFXFXFFXXXpX-5′ “AS-M128*”3′-XpFpXFXFXFXFXFXFXFXFXFXFFFFFpF-5′ “AS-M129*”3′-XpFpXFXFXFXFXFXFXFXFXFXXXFXFpX-5′ “AS-M130*”3′-XpFpXFXFXFXFXFXFXFXFXFXFXFXFpX-5′ “AS-M131*”3′-XpFpXFXFXFXFXFXFXFXFXFXFXXXXpX-5′ “AS-M132*”3′-XpFpXFXFXFXFXFXFXFXFXFXFXXXXpX-5′ “AS-M133*”3′-XpFpXFXFXFXFXFXFXFXFXFXFXFFFpF-5′ “AS-M134*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M135*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M136*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M137*”3′-XXXFXFXFXXXXXFXFXFXFXXXXXXX-5′ “AS-M138*”3′-XXXFXFXFFXXXXXXXXXXFXXXXXXX-5′ “AS-M139*”3′-XpXXFXFXFXXXXXFXFXFFFXXXXXXpX-5′ “AS-M140*”3′-XpXXFXFXFFXXXFFXFXFFFXXXXXXpX-5′ “AS-M141*”3′-XpXXFXFXFXFXFXFXFXFFFXXXXXXpX-5′ “AS-M142*”3′-XpXXFXFXFFFFFFFXFXFFFXXXXXXpX-5′ “AS-M143*”3′-XpXXFXFXFXFXFXFXFXFpXpFpXXXXXXpX-5′ “AS-M144*”3′-XXXXXXXXXXXXXXXXFXXXXXXXXXX-5′ “AS-M145*”3′-XXXXXXXXXXXXXXFXXXXXXXXXXXX-5′ “AS-M146*”3′-XXXXXXFXXXXXXXXXXXXXXXXXXXX-5′ “AS-M147*”3′-XXXXXXXXXXFXXXXXXXXXXXXXXXX-5′ “AS-M148*”3′-XXXXXXXXXXXXFXXXXXXXXXXXXXX-5′ “AS-M149*”3′-XXXXXXXXXXFXFXXXXXXXXXXXXXX-5′ “AS-M150*”3′-XXXXXXXXXXXXFXXXFXXXXXXXXXX-5′ “AS-M151*”3′-XXXXXXXXXXFXXXFXXXXXXXXXXXX-5′ “AS-M152*”3′-XXXXXXXXFXXXXXXXXXXXXXXXXXX-5′ “AS-M153*”3′-XXXXXXXXFXXXXXXXXXXXXXXXXXX-5′ “AS-M154*”3′-XXXXXXFXXXXXXXXXXXXXXXXXXXX-5′ “AS-M155*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M156*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M157*”3′-XpXXFXFFFFXXXFXXFXXFFXXXXXXpX-5′ “AS-M158*”3′-XpXXFXFFFFFFFFXXFXXFFXXXXXXpX-5′ “AS-M159*”3′-XpXXFXXXFXXXXXFXFXXFFXXXXXXpX-5′ “AS-M160*”3′-XpXXFXXXFXFXFXFXFXXFFXXXXXXpX-5′ “AS-M161*”3′-XXXXXXXXFXFXFXXXXXXXXXXXXXX-5′ “AS-M162*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M163*”3′-XXXXXXXXDXXXXXXXXXXXXXXXXXX-5′ “AS-M164*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M211”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M212”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M215”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M216”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M217”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M218”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M219”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M220”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M221”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M222”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M223”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M224”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M225”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M226”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M230”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M231”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M232”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M233”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M234”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M235”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M236”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M237”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M238”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M239”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M240”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M241”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M242”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M243”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M244”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M245”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M246”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M247”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M248”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M249”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M250”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M251”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M252”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M253”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M254”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M255”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M211*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M212*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M215*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M216*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M217*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M218*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M219*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M220*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M221*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M222*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M223*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M224*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M225*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M226*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M230*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M231*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M232*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M233*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M234*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M235*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M236*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M237*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M238*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M239*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M240*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M241*”3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ “AS-M242*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M243*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M244*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M245*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M246*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M247*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M248*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M249*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M250*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M251*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M252*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M253*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M254*”3′-XpXXXXXXXXXXXXXXXXXXXXXXXXXpX-5′ “AS-M255*” where “X” = RNA, “X” =2′-O-methyl RNA, “D” = DNA,  “F” = 2′-Fluoro NA and “p” =Phosphorothioate  linkage.

In certain additional embodiments, the antisense strand of selecteddsRNAs of the invention are extended, optionally at the 5′ end, with anexemplary 5′ extension of base “AS-M8”, “AS-M17” and “AS-M48”modification patterns respectively represented as follows:

(SEQ ID NO: 14860) 3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXU

GCU

UCGT-5′ “AS-M8, extended” (SEQ ID NO: 14860)3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXU

GCU

UCGT-5′ “AS-M17, extended” (SEQ ID NO: 14860)3′-XXXXXXXXXXXXXXXXXXXXXXXXXXXU

GCU

UCGT-5′ “AS-M48, extended” where “X” = RNA; “X” = 2′-O-methyl RNA;   “F”= 2′-Fluoro NA and “

” in bold, italics  indicates a 2′-Fluoro-adenine residue.

In certain embodiments, the sense strand of a DsiRNA of the invention ismodified—specific exemplary forms of sense strand modifications areshown below, and it is contemplated that such modified sense strands canbe substituted for the sense strand of any of the DsiRNAs shown above togenerate a DsiRNA comprising a below-depicted sense strand that annealswith an above-depicted antisense strand. Exemplary sense strandmodification patterns include:

5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM1” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM2” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM3”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM4” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM5” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM6”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM7” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM8” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM9”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM10” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM11” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM12”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM13” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM14” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM15”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM16” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM17” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM18”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM19” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM20” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM21”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM23” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM24” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM25”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM30” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM31” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM32”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM33” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM34” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM35”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM36” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM37” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM38”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM39” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM40” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM41”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM42” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM43” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM44”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM45”, “SM47”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM46” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM48” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM49”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM50” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM51” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM52”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM53” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM54” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM55”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM56” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM57” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM58”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM59” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM60” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM61”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM62” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM63” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM64”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM65” 5′-XXXXXXXXXXXXXXXXXXXXXXXpDpD-3′“SM66” 5′-XpXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM67”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM68”5′-DXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM69”5′-DpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM70”5′-DXDXXXXXXXXXXXXXXXXXXXXDD-3′ “SM71” 5′-DpXDXXXXXXXXXXXXXXXXXXXXDD-3′“SM72” 5′-XXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM73”5′-XpXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM74” 5′-DXDXXXXXXXXXDXXXXXDXXXXDD-3′“SM75” 5′-DpXDXXXXXXXXXDXXXXXDXXXXDD-3′ “SM76”5′-XpXpXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM77”5′-XpXpXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM78”5′-DpXpDXXXXXXXXXXXXXXXXXXXXDD-3′ “SM79”5′-XpXpDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM80” 5′-DXDXXXDXXXXXDXXXXXDXXXXDD-3′“SM81” 5′-DpXDXXXDXXXXXDXXXXXDXXXXpDpD-3′ “SM82” 5′C3 spacer-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM83” 5′C3 spacer-XXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM84” 5′C3 spacer-XXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM85”5′-XXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM86”5′-XpXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM87”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM88”5′-DXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM89”5′-DpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM90”5′-DXDXXXXXXXXXXXXXXXXXXXXDD-3′ “SM91” 5′-DpXDXXXXXXXXXXXXXXXXXXXXDD-3′“SM92” 5′-XXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM93”5′-XpXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM94” 5′-DXDXXXXXXXXXDXXXXXDXXXXDD-3′“SM95” 5′-DpXDXXXXXXXXXDXXXXXDXXXXDD-3′ “SM96”5′-XpXpXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM97”5′-XpXpXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM98”5′-DpXpDXXXXXXXXXXXXXXXXXXXXDD-3′ “SM99”5′-XpXpDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM100”5′-DXDXXXXXXXXXDXDXXXDDXXDDD-3′ “SM101”5′-DpXDXXXDXXXXXDXXXXXDXXXXpDpD-3′ “SM102” 5′C3 spacer-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM103” 5′C3 spacer-XXDXXXXXXXXXDXXXXXXXXXXDD-3′ “SM104” 5′C3 spacer-XXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM105”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM106” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM107” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM108”5′-XFXXXXXXXXXXXXXXXFXXXXXDD-3′ “SM110” 5′-XXXFXFXXXXXXXFXXXXXXXXXDD-3′“SM111” 5′-XFXFXFXFXXXFXFXFXFXXXXXDD-3′ “SM112”5′-XpFXFXFXFXXXFXFXFXFXXXXXpDpD-3′ “SM113”5′-XFXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM114” 5′-XXXXFFXFXXXFXFXXXXXXXXXDD-3′“SM115” 5′-XFXFXXXXXXXXXXXXFXXXXXXDD-3′ “SM116”5′-XFXFFFXFXXXFXFXFFFXXXXXDD-3′ “SM117” 5′-XFXFXFXFXXXFXFXFXFXXXXXDD-3′“SM118” 5′-XpFXFXFXFXXXFXFXFXFXXXXXpDpD-3′ “SM119”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM250” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM251” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM252”5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ “SM22” 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-35′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM120”5′-FpXFXFXFXFXFXFXFXFXFXFXXDpD-3′ “SM121”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM122”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM123”5′-FpXFXXXFXXXXXXXXXXXXXXXXpDpD-3′ “SM124”5′-FpXFXXXFXXXXXFXFXXXXXXXXpDpD-3′ “SM125”5′-FpXFXXXFXFFFXFXFXXXXXXXXpDpD-3′ “SM126”5′-FpXFXXXFXFFFXFXFXXXFFXXXpDpD-3′ “SM127”5′-FpXFXXXFXFFFXFXFXXXFFXXFpDpD-3′ “SM128”5′-FpXFXXXFXFFFXFXFXXXFFFFFpDpD-3′ “SM129”5′-FpXFXFXFXFXFXFXFXFXFXFXFpDpD-3′ “SM130”5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-FpXFXFXFXFXFXFXFXFXFXFXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-FpXFXXXFXXXXXXXXXXXXXXXXpXpX-3′ 5′-FpXFXXXFXXXXXFXFXXXXXXXXpXpX-3′5′-FpXFXXXFXFFFXFXFXXXXXXXXpXpX-3′ 5′-FpXFXXXFXFFFXFXFXXXFFXXXpXpX-3′5′-FpXFXXXFXFFFXFXFXXXFFXXFpXpX-3′ 5′-FpXFXXXFXFFFXFXFXXXFFFFFpXpX-3′5′-FpXFXFXFXFXFXFXFXFXFXFXFpXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′“SM133” 5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM134”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM135”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM136”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM137”5′-XpXXXXXXXXXXXXXXXXXXXXXXpDpD-3′ “SM138”5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXpXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM140”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM141”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM142”5′-FpXFXFXFXFXFXFXFXFXFXXXFDpD-3′ “SM143”5′-FpXFXFXFXFXFXFXFXFXFFFFFDpD-3′ “SM144”5′-FpXFXFXFXFXFXFXFXFXFXXXXDpD-3′ “SM145”5′-FpXFXFXFXFXFXFXFXFXFXFXFDpD-3′ “SM146”5′-FXFXXXFXFFFXFXFXXXXXXXXDD-3′ “SM147” 5′-FXFXXXFXFFFXFXFXXXFFXXXDD-3′“SM148” 5′-FXFXXXFXFFFXFXFXXXFFXXFDD-3′ “SM149”5′-FXFXXXFXFFFXFXFXXXFFFFFDD-3′ “SM150”5′-FpXFXFXFXFFFXFXFXFXFFXXFDpD-3′ “SM151”5′-FpXFXFXFXFFXFXXFXFXFXXXXDpD-3′ “SM152”5′-FpXFXFXXFFFFFXXFXFXXFXXXDpD-3′ “SM153”5′-ab-XXFXXFFFXXFFXXXFFFFXpXXXDD-3′ “SM154” (ab =abasic for F7 stabilization at 5′ end) 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′“SM155” 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ “SM156”5′-FpXFXXXFFFFFFFXXXFXXFXXFXpX-3′ “SM157”5′-XpXFXFXXFFFFXFXFXFXXFXXXXpX-3′ “SM158”5′-FpXFXXXFXFFXFXXFXXXFXXXXXpX-3′ “SM159”5′-FpXFXFXFXFFFXFXFXFXFXXXXXX-3′ “SM160”5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-FpXFXFXFXFXFXFXFXFXFXXXFXpX-3′5′-FpXFXFXFXFXFXFXFXFXFFFFFXpX-3′ 5′-FpXFXFXFXFXFXFXFXFXFXXXXXpX-3′5′-FpXFXFXFXFXFXFXFXFXFXFXFXpX-3′ 5′-FXFXXXFXFFFXFXFXXXXXXXXXX-3′5′-FXFXXXFXFFFXFXFXXXFFXXXXX-3′ 5′-FXFXXXFXFFFXFXFXXXFFXXFXX-3′5′-FXFXXXFXFFFXFXFXXXFFFFFXX-3′ 5′-FpXFXFXFXFFFXFXFXFXFFXXFXpX-3′5′-FpXFXFXFXFFXFXXFXFXFXXXXXpX-3′ 5′-FpXFXFXXFFFFFXXFXFXXFXXXXpX-3′5′-ab-XXFXXFFFXXFFXXXFFFFXpXXXXX-3′ (ab = abasic for F7 stabilizationat 5′ end) 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-FpXFXXXFFFFFFFXXXFXXFXXFXpX-3′5′-XpXFXFXXFFFFXFXFXFXXFXXXXpX-3′ 5′-FpXFXXXFXFFXFXXFXXXFXXXXXpX-3′5′-FpXFXFXFXFFFXFXFXFXFXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM253”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM255” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM256” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM257”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM258” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM259” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM260”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM261” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM262” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM263”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM264” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM265” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM266”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM267” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM268” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM269”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM270” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM271” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM275”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM276” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM277” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM278”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM279” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM280” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM281”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM282” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM283” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM284”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM285” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM286” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM287”5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM288” 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′“SM289” 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ “SM300”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM301”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM302”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM303”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM304”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM305”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM306”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM307”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM308”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM309”5′-XpXXXXXXXXXXXXXXXXXXXXXXDpD-3′ “SM310”5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ 5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′5′-XpXXXXXXXXXXXXXXXXXXXXXXXpX-3′ where “X” = RNA, “X” =2′-O-methyl RNA, “D” = DNA, “F” = 2′-Fluoro NA and “p” =  Phosphorothioate linkage.

It is contemplated that in certain embodiments of the invention, for all2′-O-methyl modification patterns disclosed herein, any or all sites of2′-O-methyl modification can optionally be replaced by a 2′-Fluoromodification. The above modification patterns can also be incorporatedinto, e.g., the extended DsiRNA structures and mismatch and/or frayedDsiRNA structures described below.

In another embodiment, the DsiRNA comprises strands having equal lengthspossessing 1-3 mismatched residues that serve to orient Dicer cleavage(specifically, one or more of positions 1, 2 or 3 on the first strand ofthe DsiRNA, when numbering from the 3′-terminal residue, are mismatchedwith corresponding residues of the 5′-terminal region on the secondstrand when first and second strands are annealed to one another). Anexemplary 27 mer DsiRNA agent with two terminal mismatched residues isshown:

wherein “X”, RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed. Any of the residues of such agents can optionallybe 2′-O-methyl RNA monomers—alternating positioning of 2′-O-methyl RNAmonomers that commences from the 3′-terminal residue of the bottom(second) strand, as shown for above asymmetric agents, can also be usedin the above “blunt/fray” DsiRNA agent. In one embodiment, the topstrand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In certain additional embodiments, the present invention providescompositions for RNA interference (RNAi) that possess one or more basepaired deoxyribonucleotides within a region of a double strandedribonucleic acid (dsRNA) that is positioned 3′ of a projected sensestrand Dicer cleavage site and correspondingly 5′ of a projectedantisense strand Dicer cleavage site. The compositions of the inventioncomprise a dsRNA which is a precursor molecule, i.e., the dsRNA of thepresent invention is processed in vivo to produce an active smallinterfering nucleic acid (siRNA). The dsRNA is processed by Dicer to anactive siRNA which is incorporated into RISC.

In certain embodiments, the DsiRNA agents of the invention can have thefollowing exemplary structures (noting that any of the followingexemplary structures can be combined, e.g., with the bottom strandmodification patterns of the above-described structures—in one specificexample, the bottom strand modification pattern shown in any of theabove structures is applied to the 27 most 3′ residues of the bottomstrand of any of the following structures; in another specific example,the bottom strand modification pattern shown in any of the abovestructures upon the 23 most 3′ residues of the bottom strand is appliedto the 23 most 3′ residues of the bottom strand of any of the followingstructures):

In one such embodiment, the DsiRNA comprises the following (an exemplary“right-extended”, “DNA extended” DsiRNA):

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)XX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)D_(N)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-8 or 1-10.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXX_(N*)[X1/D1]_(N)DD-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXX_(N*)[X2/D2]_(N)ZZ-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA or RNA,and “N”=1 to 50 or more, but is optionally 1-8 or 1-10, where at leastone D1_(N) is present in the top strand and is base paired with acorresponding D2_(N) in the bottom strand. Optionally, D1_(N) andD1_(N+1) are base paired with corresponding D2_(N) and D2_(N+1); D1_(N),D1_(N+1) and D1_(N+2) are base paired with corresponding D2_(N),D1_(N+1) and D1_(N+2), etc. “N*”=0 to 15 or more, but is optionally 0,1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In the structures depicted herein, the 5′ end of either the sense strandor antisense strand can optionally comprise a phosphate group.

In another embodiment, a DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another). An exemplary DNA:DNA-extended DsiRNA agent with twoterminal mismatched residues is shown:

wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-15 or, optionally, 1-8. “N*”=0 to 15 or more, but is optionally 0, 1,2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be2′-O-methyl RNA monomers—alternating positioning of 2′-O-methyl RNAmonomers that commences from the 3′-terminal residue of the bottom(second) strand, as shown for above asymmetric agents, can also be usedin the above “blunt/fray” DsiRNA agent. In one embodiment, the topstrand (first strand) is the sense strand, and the bottom strand (secondstrand) is the antisense strand. Alternatively, the bottom strand is thesense strand and the top strand is the antisense strand. Modificationand DNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In one embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsRNA structure. Anexemplary structure for such a molecule is shown:

5′-XXXXXXXXXXXXXXXXXXXDDXX-3′ 3′-YXXXXXXXXXXXXXXXXXDDXXXX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. The above structureis modeled to force Dicer to cleave a minimum of a 21 mer duplex as itsprimary post-processing form. In embodiments where the bottom strand ofthe above structure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand will help reduce off-target effects(as prior studies have shown a 2′-O-methyl modification of at least thepenultimate position from the 5′ terminus of the antisense strand toreduce off-target effects; see, e.g., US 2007/0223427).

In one embodiment, the DsiRNA comprises the following (an exemplary“left-extended”, “DNA extended” DsiRNA):

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)XX-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. “Z”=DNA or RNA. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, 5 or 6. “Z”=DNA or RNA. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)ZZXXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strandis the sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-D_(N)ZZXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′ 3′-D_(N)XXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-8 or 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, 5 or 6. “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises:

5′-[X1/D1]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-[X2/D2]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)ZZ-5′wherein “X”=RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1 to 50 or more, butis optionally 1-8 or 1-10, where at least one D1_(N) is present in thetop strand and is base paired with a corresponding D2_(N) in the bottomstrand. Optionally, D1_(N) and D1_(N+1) are base paired withcorresponding D2_(N) and D2_(N+1); D1_(N), D1_(N+1) and D1_(N+2) arebase paired with corresponding D2_(N), D1_(N+1) and D1_(N+2), etc.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In a related embodiment, the DsiRNA comprises:

5′-[X1/D1]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′3′-[X2/D2]_(N)XXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “D”=DNA, “Y” is an optional overhang domain comprisedof 0-10 RNA monomers that are optionally 2′-O-methyl RNA monomers—incertain embodiments, “Y” is an overhang domain comprised of 1-4 RNAmonomers that are optionally 2′-O-methyl RNA monomers, and “N”=1 to 50or more, but is optionally 1-8 or 1-10, where at least one D1_(N) ispresent in the top strand and is base paired with a corresponding D2_(N)in the bottom strand. Optionally, D1_(N) and D1_(N+1) are base pairedwith corresponding D2_(N) and D2_(N+1); D1_(N), D1_(N+1) and D1_(N+2)are base paired with corresponding D2_(N), D1_(N+1) and D1_(N+2), etc.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another embodiment, the DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another). An exemplary DNA:DNA-extended DsiRNA agent with twoterminal mismatched residues is shown:

wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-8 or 1-10. “N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or6. Any of the residues of such agents can optionally be 2′-O-methyl RNAmonomers—alternating positioning of 2′-O-methyl RNA monomers thatcommences from the 3′-terminal residue of the bottom (second) strand, asshown for above asymmetric agents, can also be used in the above“blunt/fray” DsiRNA agent. In one embodiment, the top strand (firststrand) is the sense strand, and the bottom strand (second strand) isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. Modification andDNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In another embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsRNA structure.Exemplary structures for such a molecule are shown:

5′-XXDDXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′ 3′-DDXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′or 5′-XDXDXXXXXXXXXXXXXXXXXXXX_(N*)Y-3′3′-DXDXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In any of the above embodiments where the bottom strand of the abovestructure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand will help reduce off-target effects(as prior studies have shown a 2′-O-methyl modification of at least thepenultimate position from the 5′ terminus of the antisense strand toreduce off-target effects; see, e.g., US 2007/0223427).

In certain embodiments, the “D” residues of the above structures includeat least one PS-DNA or PS-RNA. Optionally, the “D” residues of the abovestructures include at least one modified nucleotide that inhibits Dicercleavage.

While the above-described “DNA-extended” DsiRNA agents can becategorized as either “left extended” or “right extended”, DsiRNA agentscomprising both left- and right-extended DNA-containing sequences withina single agent (e.g., both flanks surrounding a core dsRNA structure aredsDNA extensions) can also be generated and used in similar manner tothose described herein for “right-extended” and “left-extended” agents.

In some embodiments, the DsiRNA of the instant invention furthercomprises a linking moiety or domain that joins the sense and antisensestrands of a DNA:DNA-extended DsiRNA agent. Optionally, such a linkingmoiety domain joins the 3′ end of the sense strand and the 5′ end of theantisense strand. The linking moiety may be a chemical (non-nucleotide)linker, such as an oligomethylenediol linker, oligoethylene glycollinker, or other art-recognized linker moiety. Alternatively, the linkercan be a nucleotide linker, optionally including an extended loop and/ortetraloop.

In one embodiment, the DsiRNA agent has an asymmetric structure, withthe sense strand having a 25-base pair length, and the antisense strandhaving a 27-base pair length with a 1-4 base 3′-overhang (e.g., a onebase 3′-overhang, a two base 3′-overhang, a three base 3′-overhang or afour base 3′-overhang). In another embodiment, this DsiRNA agent has anasymmetric structure further containing 2 deoxynucleotides at the 3′ endof the sense strand.

In another embodiment, the DsiRNA agent has an asymmetric structure,with the antisense strand having a 25-base pair length, and the sensestrand having a 27-base pair length with a 1-4 base 3′-overhang (e.g., aone base 3′-overhang, a two base 3′-overhang, a three base 3′-overhangor a four base 3′-overhang). In another embodiment, this DsiRNA agenthas an asymmetric structure further containing 2 deoxyribonucleotides atthe 3′ end of the antisense strand.

Exemplary Glycolate Oxidase targeting DsiRNA agents of the invention,and their associated Glycolate Oxidase target sequences, include thefollowing, presented in the below series of tables:

Table Number: (2) Selected Human Anti-Glycolate Oxidase DsiRNA Agents(Asymmetrics); (3) Selected Human Anti-Glycolate Oxidase DsiRNAs,Unmodified Duplexes (Asymmetrics);

(4) DsiRNA Target Sequences (21 mers) in Glycolate Oxidase mRNA;

(5) Selected Human Anti-Glycolate Oxidase “Blunt/Blunt” DsiRNAs; and

(6) DsiRNA Component 19 Nucleotide Target Sequences in Glycolate OxidasemRNA(7) Human Anti-Glycolate Oxidase DsiRNAs Predicted to have >50%Knockdown Efficacy (Asymmetrics);(8) DsiRNA Target Sequences (21 mers) of Human Anti-Glycolate OxidaseDsiRNAs Predicted to have >50% Knockdown Efficacy;(9) “Blunt/Blunt” DsiRNAs Corresponding to Human Anti-Glycolate OxidaseDsiRNAs Predicted to have >50% Knockdown Efficacy; and(10) DsiRNA Component 19 Nucleotide Target Sequences of HumanAnti-Glycolate Oxidase DsiRNAs Predicted to have >50% Knockdown Efficacy

Lengthy table referenced here US20170306333A1-20171026-T00001 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00002 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00003 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00004 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00005 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00006 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00007 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00008 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20170306333A1-20171026-T00009 Pleaserefer to the end of the specification for access instructions.

Within Tables 2, 3, 5, 7 and 9 above, underlined residues indicate2′-O-methyl residues, UPPER CASE indicates ribonucleotides, and lowercase denotes deoxyribonucleotides. The DsiRNA agents of Tables 2, 3 and7 above are 25/27 mer agents possessing a blunt end. The structuresand/or modification patterning of the agents of Tables 2, 3 and 7 abovecan be readily adapted to the above generic sequence structures, e.g.,the 3′ overhang of the second strand can be extended or contracted,2′-O-methylation of the second strand can be expanded towards the 5′ endof the second strand, optionally at alternating sites, etc. Such furthermodifications are optional, as 25/27 mer DsiRNAs with such modificationscan also be readily designed from the above DsiRNA agents and are alsoexpected to be functional inhibitors of Glycolate Oxidase expression.Similarly, the 27 mer “blunt/blunt” DsiRNA structures and/ormodification patterns of the agents of Tables 5 and 9 above can also bereadily adapted to the above generic sequence structures, e.g., forapplication of modification patterning of the antisense strand to suchstructures and/or adaptation of such sequences to the above genericstructures.

In certain embodiments, 27 mer DsiRNAs possessing independent strandlengths each of 27 nucleotides are designed and synthesized fortargeting of the same sites within the Glycolate Oxidase transcript asthe asymmetric “25/27” structures shown in Tables 2, 3 and 7 herein.Exemplary “27/27” DsiRNAs are optionally designed with a “blunt/blunt”structure as shown for the DsiRNAs of Tables 5 and 9 above.

In certain embodiments, the dsRNA agents of the invention require, e.g.,at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25 or at least 26 residues of the first strand to be complementary tocorresponding residues of the second strand. In certain relatedembodiments, these first strand residues complementary to correspondingresidues of the second strand are optionally consecutive residues.

As used herein “DsiRNAmm” refers to a DisRNA having a “mismatch tolerantregion” containing one, two, three or four mismatched base pairs of theduplex formed by the sense and antisense strands of the DsiRNA, wheresuch mismatches are positioned within the DsiRNA at a location(s) lyingbetween (and thus not including) the two terminal base pairs of eitherend of the DsiRNA. The mismatched base pairs are located within a“mismatch-tolerant region” which is defined herein with respect to thelocation of the projected Ago2 cut site of the corresponding targetnucleic acid. The mismatch tolerant region is located “upstream of” theprojected Ago2 cut site of the target strand. “Upstream” in this contextwill be understood as the 5′-most portion of the DsiRNAmm duplex, where5′ refers to the orientation of the sense strand of the DsiRNA duplex.Therefore, the mismatch tolerant region is upstream of the base on thesense (passenger) strand that corresponds to the projected Ago2 cut siteof the target nucleic acid (see FIG. 1); alternatively, when referringto the antisense (guide) strand of the DsiRNAmm, the mismatch tolerantregion can also be described as positioned downstream of the base thatis complementary to the projected Ago2 cut site of the target nucleicacid, that is, the 3′-most portion of the antisense strand of theDsiRNAmm (where position 1 of the antisense strand is the 5′ terminalnucleotide of the antisense strand, see FIG. 1).

In one embodiment, for example with numbering as depicted in FIG. 1, themismatch tolerant region is positioned between and including base pairs3-9 when numbered from the nucleotide starting at the 5′ end of thesense strand of the duplex. Therefore, a DsiRNAmm of the inventionpossesses a single mismatched base pair at any one of positions 3, 4, 5,6, 7, 8 or 9 of the sense strand of a right-hand extended DsiRNA (whereposition 1 is the 5′ terminal nucleotide of the sense strand andposition 9 is the nucleotide residue of the sense strand that isimmediately 5′ of the projected Ago2 cut site of the target GlycolateOxidase RNA sequence corresponding to the sense strand sequence). Incertain embodiments, for a DsiRNAmm that possesses a mismatched basepair nucleotide at any of positions 3, 4, 5, 6, 7, 8 or 9 of the sensestrand, the corresponding mismatched base pair nucleotide of theantisense strand not only forms a mismatched base pair with the DsiRNAmmsense strand sequence, but also forms a mismatched base pair with aDsiRNAmm target Glycolate Oxidase RNA sequence (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, andcomplementarity is similarly disrupted between the antisense strandsequence of the DsiRNAmm and the target Glycolate Oxidase RNA sequence).In alternative embodiments, the mismatch base pair nucleotide of theantisense strand of a DsiRNAmm only form a mismatched base pair with acorresponding nucleotide of the sense strand sequence of the DsiRNAmm,yet base pairs with its corresponding target Glycolate Oxidase RNAsequence nucleotide (thus, complementarity between the antisense strandsequence and the sense strand sequence is disrupted at the mismatchedbase pair within the DsiRNAmm, yet complementarity is maintained betweenthe antisense strand sequence of the DsiRNAmm and the target GlycolateOxidase RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region (mismatch region) as described above(e.g., a DsiRNAmm harboring a mismatched nucleotide residue at any oneof positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand) can furtherinclude one, two or even three additional mismatched base pairs. Inpreferred embodiments, these one, two or three additional mismatchedbase pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and/or9 of the sense strand (and at corresponding residues of the antisensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of the sensestrand can occur, e.g., at nucleotides of both position 4 and position 6of the sense strand (with mismatch also occurring at correspondingnucleotide residues of the antisense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that base pair with theantisense strand sequence (e.g., for a DsiRNAmm possessing mismatchednucleotides at positions 3 and 6, but not at positions 4 and 5, themismatched residues of sense strand positions 3 and 6 are interspersedby two nucleotides that form matched base pairs with correspondingresidues of the antisense strand). For example, two residues of thesense strand (located within the mismatch-tolerant region of the sensestrand) that form mismatched base pairs with the corresponding antisensestrand sequence can occur with zero, one, two, three, four or fivematched base pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 4 and 8, but not at positions 5,6 and 7, the mismatched residues of sense strand positions 3 and 4 areadjacent to one another, while the mismatched residues of sense strandpositions 4 and 8 are interspersed by three nucleotides that formmatched base pairs with corresponding residues of the antisense strand).For example, three residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two, three or four matched base pairs located between any twoof these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of thesense strand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 5, 7 and 8, but not at positions4 and 6, the mismatched residues of sense strand positions 7 and 8 areadjacent to one another, while the mismatched residues of sense strandpositions 3 and 5 are interspersed by one nucleotide that forms amatched base pair with the corresponding residue of the antisensestrand—similarly, the mismatched residues of sense strand positions 5and 7 are also interspersed by one nucleotide that forms a matched basepair with the corresponding residue of the antisense strand). Forexample, four residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two or three matched base pairs located between any two ofthese mismatched base pairs.

In another embodiment, for example with numbering also as depicted inFIG. 1, a DsiRNAmm of the invention comprises a mismatch tolerant regionwhich possesses a single mismatched base pair nucleotide at any one ofpositions 17, 18, 19, 20, 21, 22 or 23 of the antisense strand of theDsiRNA (where position 1 is the 5′ terminal nucleotide of the antisensestrand and position 17 is the nucleotide residue of the antisense strandthat is immediately 3′ (downstream) in the antisense strand of theprojected Ago2 cut site of the target Glycolate Oxidase RNA sequencesufficiently complementary to the antisense strand sequence). In certainembodiments, for a DsiRNAmm that possesses a mismatched base pairnucleotide at any of positions 17, 18, 19, 20, 21, 22 or 23 of theantisense strand with respect to the sense strand of the DsiRNAmm, themismatched base pair nucleotide of the antisense strand not only forms amismatched base pair with the DsiRNAmm sense strand sequence, but alsoforms a mismatched base pair with a DsiRNAmm target Glycolate OxidaseRNA sequence (thus, complementarity between the antisense strandsequence and the sense strand sequence is disrupted at the mismatchedbase pair within the DsiRNAmm, and complementarity is similarlydisrupted between the antisense strand sequence of the DsiRNAmm and thetarget Glycolate Oxidase RNA sequence). In alternative embodiments, themismatch base pair nucleotide of the antisense strand of a DsiRNAmm onlyforms a mismatched base pair with a corresponding nucleotide of thesense strand sequence of the DsiRNAmm, yet base pairs with itscorresponding target Glycolate Oxidase RNA sequence nucleotide (thus,complementarity between the antisense strand sequence and the sensestrand sequence is disrupted at the mismatched base pair within theDsiRNAmm, yet complementarity is maintained between the antisense strandsequence of the DsiRNAmm and the target Glycolate Oxidase RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 17, 18, 19, 20,21, 22 or 23 of the antisense strand) can further include one, two oreven three additional mismatched base pairs. In preferred embodiments,these one, two or three additional mismatched base pairs of the DsiRNAmmoccur at position(s) 17, 18, 19, 20, 21, 22 and/or 23 of the antisensestrand (and at corresponding residues of the sense strand). In oneembodiment where one additional mismatched base pair is present within aDsiRNAmm, the two mismatched base pairs of the antisense strand canoccur, e.g., at nucleotides of both position 18 and position 20 of theantisense strand (with mismatch also occurring at correspondingnucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 17 and 20, but not at positions 18 and 19, the mismatchedresidues of antisense strand positions 17 and 20 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 17, 18 and 22, but not at positions19, 20 and 21, the mismatched residues of antisense strand positions 17and 18 are adjacent to one another, while the mismatched residues ofantisense strand positions 18 and 122 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 18, 20, 22 and 23, but not atpositions 19 and 21, the mismatched residues of antisense strandpositions 22 and 23 are adjacent to one another, while the mismatchedresidues of antisense strand positions 18 and 20 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the mismatched residues of antisensestrand positions 20 and 22 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

For reasons of clarity, the location(s) of mismatched nucleotideresidues within the above DsiRNAmm agents are numbered in reference tothe 5′ terminal residue of either sense or antisense strands of theDsiRNAmm. The numbering of positions located within themismatch-tolerant region (mismatch region) of the antisense strand canshift with variations in the proximity of the 5′ terminus of the senseor antisense strand to the projected Ago2 cleavage site. Thus, thelocation(s) of preferred mismatch sites within either antisense strandor sense strand can also be identified as the permissible proximity ofsuch mismatches to the projected Ago2 cut site. Accordingly, in onepreferred embodiment, the position of a mismatch nucleotide of the sensestrand of a DsiRNAmm is the nucleotide residue of the sense strand thatis located immediately 5′ (upstream) of the projected Ago2 cleavage siteof the corresponding target Glycolate Oxidase RNA sequence. In otherpreferred embodiments, a mismatch nucleotide of the sense strand of aDsiRNAmm is positioned at the nucleotide residue of the sense strandthat is located two nucleotides 5′ (upstream) of the projected Ago2cleavage site, three nucleotides 5′ (upstream) of the projected Ago2cleavage site, four nucleotides 5′ (upstream) of the projected Ago2cleavage site, five nucleotides 5′ (upstream) of the projected Ago2cleavage site, six nucleotides 5′ (upstream) of the projected Ago2cleavage site, seven nucleotides 5′ (upstream) of the projected Ago2cleavage site, eight nucleotides 5′ (upstream) of the projected Ago2cleavage site, or nine nucleotides 5′ (upstream) of the projected Ago2cleavage site.

Exemplary single mismatch-containing 25/27 mer DsiRNAs (DsiRNAmm)include the following structures (such mismatch-containing structuresmay also be incorporated into other exemplary DsiRNA structures shownherein).

5′-XX^(M)XXXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXX_(M)XXXXXXXXXXXXXXXXXXXXXX-5′5′-XXX^(M)XXXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXX_(M)XXXXXXXXXXXXXXXXXXXXX-5′5′-XXXX^(M)XXXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXX_(M)XXXXXXXXXXXXXXXXXXXX-5′5′-XXXXX^(M)XXXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXX_(M)XXXXXXXXXXXXXXXXXXX-5′5′-XXXXXX^(M)XXXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXX_(M)XXXXXXXXXXXXXXXXXX-5′5′-XXXXXXX^(M)XXXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXX_(M)XXXXXXXXXXXXXXXXX-5′5′-XXXXXXXX^(M)XXXXXXXXXXXXXXDD-3′ 3′-XXXXXXXXXX_(M)XXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “D”=DNA and “M”=Nucleic acid residues (RNA, DNA ornon-natural or modified nucleic acids) that do not base pair (hydrogenbond) with corresponding “M” residues of otherwise complementary strandwhen strands are annealed. Any of the residues of such agents canoptionally be 2′-O-methyl RNA monomers—alternating positioning of2′-O-methyl RNA monomers that commences from the 3′-terminal residue ofthe bottom (second) strand, as shown above, can also be used in theabove DsiRNAmm agents. For the above mismatch structures, the top strandis the sense strand, and the bottom strand is the antisense strand.

In certain embodiments, a DsiRNA of the invention can contain mismatchesthat exist in reference to the target Glycolate Oxidase RNA sequence yetdo not necessarily exist as mismatched base pairs within the two strandsof the DsiRNA—thus, a DsiRNA can possess perfect complementarity betweenfirst and second strands of a DsiRNA, yet still possess mismatchedresidues in reference to a target Glycolate Oxidase RNA (which, incertain embodiments, may be advantageous in promoting efficacy and/orpotency and/or duration of effect). In certain embodiments, wheremismatches occur between antisense strand and target Glycolate OxidaseRNA sequence, the position of a mismatch is located within the antisensestrand at a position(s) that corresponds to a sequence of the sensestrand located 5′ of the projected Ago2 cut site of the targetregion—e.g., antisense strand residue(s) positioned within the antisensestrand to the 3′ of the antisense residue which is complementary to theprojected Ago2 cut site of the target sequence.

Exemplary 25/27 mer DsiRNAs that harbor a single mismatched residue inreference to target sequences include the following structures.

Target RNA Sequence: 5′-...AXXXXXXXXXXXXXXXXXXXX...-3′ DsiRNAmm SenseStrand: 5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-EXXXXXXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XAXXXXXXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XEXXXXXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...AXXXXXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-BXXXXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXEXXXXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XAXXXXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XBXXXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXEXXXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXAXXXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXBXXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXEXXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXAXXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXBXXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXEXXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXXAXXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXBXXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXXEXXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXXXAXXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXXBXXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXXXEXXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXXXXAXXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXXXBXXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXXXXEXXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXXXXXAXXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXXXXBXXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXXXXXEXXXXXXXXXXXXXXXXX-5′ Target RNA Sequence:5′-...XXXXXXXXAXXXXXXXXXX...-3′ DsiRNAmm Sense Strand:5′-XXXXXXXXBXXXXXXXXXXXXXXDD-3′ DsiRNAmm Antisense Strand:3′-XXXXXXXXXXEXXXXXXXXXXXXXXXX-5′wherein “X”, RNA, “D”=DNA and “E”=Nucleic acid residues (RNA, DNA ornon-natural or modified nucleic acids) that do not base pair (hydrogenbond) with corresponding “A” RNA residues of otherwise complementary(target) strand when strands are annealed, yet optionally do base pairwith corresponding “B” residues (“B” residues are also RNA, DNA ornon-natural or modified nucleic acids). Any of the residues of suchagents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown above, canalso be used in the above DsiRNA agents.

In certain embodiments, the guide strand of a dsRNA of the inventionthat is sufficiently complementary to a target RNA (e.g., mRNA) along atleast 19 nucleotides of the target gene sequence to reduce target geneexpression is not perfectly complementary to the at least 19 nucleotidelong target gene sequence. Rather, it is appreciated that the guidestrand of a dsRNA of the invention that is sufficiently complementary toa target mRNA along at least 19 nucleotides of a target RNA sequence toreduce target gene expression can have one, two, three, or even four ormore nucleotides that are mismatched with the 19 nucleotide or longertarget strand sequence. Thus, for a 19 nucleotide target RNA sequence,the guide strand of a dsRNA of the invention can be sufficientlycomplementary to the target RNA sequence to reduce target gene levelswhile possessing, e.g., only 15/19, 16/19, 17/19 or 18/19 matchednucleotide residues between guide strand and target RNA sequence.

In addition to the above-exemplified structures, dsRNAs of the inventioncan also possess one, two or three additional residues that form furthermismatches with the target Glycolate Oxidase RNA sequence. Suchmismatches can be consecutive, or can be interspersed by nucleotidesthat form matched base pairs with the target Glycolate Oxidase RNAsequence. Where interspersed by nucleotides that form matched basepairs, mismatched residues can be spaced apart from each other within asingle strand at an interval of one, two, three, four, five, six, sevenor even eight base paired nucleotides between such mismatch-formingresidues.

As for the above-described DsiRNAmm agents, a preferred location withindsRNAs (e.g., DsiRNAs) for antisense strand nucleotides that formmismatched base pairs with target Glycolate Oxidase RNA sequence (yetmay or may not form mismatches with corresponding sense strandnucleotides) is within the antisense strand region that is located 3′(downstream) of the antisense strand sequence which is complementary tothe projected Ago2 cut site of the DsiRNA (e.g., in FIG. 1, the regionof the antisense strand which is 3′ of the projected Ago2 cut site ispreferred for mismatch-forming residues and happens to be located atpositions 17-23 of the antisense strand for the 25/27 mer agent shown inFIG. 1). Thus, in one embodiment, the position of a mismatch nucleotide(in relation to the target Glycolate Oxidase RNA sequence) of theantisense strand of a DsiRNAmm is the nucleotide residue of theantisense strand that is located immediately 3′ (downstream) within theantisense strand sequence of the projected Ago2 cleavage site of thecorresponding target Glycolate Oxidase RNA sequence. In other preferredembodiments, a mismatch nucleotide of the antisense strand of a DsiRNAmm(in relation to the target Glycolate Oxidase RNA sequence) is positionedat the nucleotide residue of the antisense strand that is located twonucleotides 3′ (downstream) of the corresponding projected Ago2 cleavagesite, three nucleotides 3′ (downstream) of the corresponding projectedAgo2 cleavage site, four nucleotides 3′ (downstream) of thecorresponding projected Ago2 cleavage site, five nucleotides 3′(downstream) of the corresponding projected Ago2 cleavage site, sixnucleotides 3′ (downstream) of the projected Ago2 cleavage site, sevennucleotides 3′ (downstream) of the projected Ago2 cleavage site, eightnucleotides 3′ (downstream) of the projected Ago2 cleavage site, or ninenucleotides 3′ (downstream) of the projected Ago2 cleavage site.

In dsRNA agents possessing two mismatch-forming nucleotides of theantisense strand (where mismatch-forming nucleotides are mismatchforming in relation to target Glycolate Oxidase RNA sequence),mismatches can occur consecutively (e.g., at consecutive positions alongthe antisense strand nucleotide sequence). Alternatively, nucleotides ofthe antisense strand that form mismatched base pairs with the targetGlycolate Oxidase RNA sequence can be interspersed by nucleotides thatbase pair with the target Glycolate Oxidase RNA sequence (e.g., for aDsiRNA possessing mismatch-forming nucleotides at positions 17 and 20(starting from the 5′ terminus (position 1) of the antisense strand ofthe 25/27 mer agent shown in FIG. 1), but not at positions 18 and 19,the mismatched residues of sense strand positions 17 and 20 areinterspersed by two nucleotides that form matched base pairs withcorresponding residues of the target Glycolate Oxidase RNA sequence).For example, two residues of the antisense strand (located within themismatch-tolerant region of the antisense strand) that form mismatchedbase pairs with the corresponding target Glycolate Oxidase RNA sequencecan occur with zero, one, two, three, four or five matched base pairs(with respect to target Glycolate Oxidase RNA sequence) located betweenthese mismatch-forming base pairs.

For certain dsRNAs possessing three mismatch-forming base pairs(mismatch-forming with respect to target Glycolate Oxidase RNAsequence), mismatch-forming nucleotides can occur consecutively (e.g.,in a triplet along the antisense strand nucleotide sequence).Alternatively, nucleotides of the antisense strand that form mismatchedbase pairs with the target Glycolate Oxidase RNA sequence can beinterspersed by nucleotides that form matched base pairs with the targetGlycolate Oxidase RNA sequence (e.g., for a DsiRNA possessing mismatchednucleotides at positions 17, 18 and 22, but not at positions 19, 20 and21, the mismatch-forming residues of antisense strand positions 17 and18 are adjacent to one another, while the mismatch-forming residues ofantisense strand positions 18 and 22 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe target Glycolate Oxidase RNA). For example, three residues of theantisense strand (located within the mismatch-tolerant region of theantisense strand) that form mismatched base pairs with the correspondingtarget Glycolate Oxidase RNA sequence can occur with zero, one, two,three or four matched base pairs located between any two of thesemismatch-forming base pairs.

For certain dsRNAs possessing four mismatch-forming base pairs(mismatch-forming with respect to target Glycolate Oxidase RNAsequence), mismatch-forming nucleotides can occur consecutively (e.g.,in a quadruplet along the sense strand nucleotide sequence).Alternatively, nucleotides of the antisense strand that form mismatchedbase pairs with the target Glycolate Oxidase RNA sequence can beinterspersed by nucleotides that form matched base pairs with the targetGlycolate Oxidase RNA sequence (e.g., for a DsiRNA possessingmismatch-forming nucleotides at positions 17, 19, 21 and 22, but not atpositions 18 and 20, the mismatch-forming residues of antisense strandpositions 21 and 22 are adjacent to one another, while themismatch-forming residues of antisense strand positions 17 and 19 areinterspersed by one nucleotide that forms a matched base pair with thecorresponding residue of the target Glycolate Oxidase RNAsequence—similarly, the mismatch-forming residues of antisense strandpositions 19 and 21 are also interspersed by one nucleotide that forms amatched base pair with the corresponding residue of the target GlycolateOxidase RNA sequence). For example, four residues of the antisensestrand (located within the mismatch-tolerant region of the antisensestrand) that form mismatched base pairs with the corresponding targetGlycolate Oxidase RNA sequence can occur with zero, one, two or threematched base pairs located between any two of these mismatch-formingbase pairs.

The above DsiRNAmm and other dsRNA structures are described in order toexemplify certain structures of DsiRNAmm and dsRNA agents. Design of theabove DsiRNAmm and dsRNA structures can be adapted to generate, e.g.,DsiRNAmm forms of other DsiRNA structures shown infra. As exemplifiedabove, dsRNAs can also be designed that possess single mismatches (ortwo, three or four mismatches) between the antisense strand of the dsRNAand a target sequence, yet optionally can retain perfect complementaritybetween sense and antisense strand sequences of a dsRNA.

It is further noted that the dsRNA agents exemplified infra can alsopossess insertion/deletion (in/del) structures within theirdouble-stranded and/or target Glycolate Oxidase RNA-aligned structures.Accordingly, the dsRNAs of the invention can be designed to possessin/del variations in, e.g., antisense strand sequence as compared totarget Glycolate Oxidase RNA sequence and/or antisense strand sequenceas compared to sense strand sequence, with preferred location(s) forplacement of such in/del nucleotides corresponding to those locationsdescribed above for positioning of mismatched and/or mismatch-formingbase pairs.

It is also noted that the DsiRNAs of the instant invention can toleratemismatches within the 3′-terminal region of the sense strand/5′-terminalregion of the antisense strand, as this region is modeled to beprocessed by Dicer and liberated from the guide strand sequence thatloads into RISC. Exemplary DsiRNA structures of the invention thatharbor such mismatches include the following:

Target RNA Sequence: 5′-...XXXXXXXXXXXXXXXXXXXXXHXXX...-3′ DsiRNA SenseStrand: 5′-XXXXXXXXXXXXXXXXXXXXXIXDD-3′ DsiRNA Antisense Strand:3′-XXXXXXXXXXXXXXXXXXXXXXXJXXX-5′ Target RNA Sequence:5′-...XXXXXXXXXXXXXXXXXXXXXXHXX...-3′ DsiRNA Sense Strand:5′-XXXXXXXXXXXXXXXXXXXXXXIDD-3′ DsiRNA Antisense Strand:3′-XXXXXXXXXXXXXXXXXXXXXXXXJXX-5′ Target RNA Sequence:5′-...XXXXXXXXXXXXXXXXXXXXXXXHX...-3′ DsiRNA Sense Strand:5′-XXXXXXXXXXXXXXXXXXXXXXXID-3′ DsiRNA Antisense Strand:3′-XXXXXXXXXXXXXXXXXXXXXXXXXJX-5′ Target RNA Sequence:5′-...XXXXXXXXXXXXXXXXXXXXXXXXH...-3′ DsiRNA Sense Strand:5′-XXXXXXXXXXXXXXXXXXXXXXXDI-3′ DsiRNA Antisense Strand:3′-XXXXXXXXXXXXXXXXXXXXXXXXXXJ-5′wherein “X”=RNA, “D”=DNA and “I” and “J”=Nucleic acid residues (RNA, DNAor non-natural or modified nucleic acids) that do not base pair(hydrogen bond) with one another, yet optionally “J” is complementary totarget RNA sequence nucleotide “H”. Any of the residues of such agentscan optionally be 2′-O-methyl RNA monomers—alternating positioning of2′-O-methyl RNA monomers that commences from the 3′-terminal residue ofthe bottom (second) strand, as shown above—or any of the above-describedmethylation patterns—can also be used in the above DsiRNA agents. Theabove mismatches can also be combined within the DsiRNAs of the instantinvention.

In the below structures, such mismatches are introduced within theasymmetric

HAO1-1171 DsiRNA (newly-introduced mismatch residues are italicized):HAO1-1171 25/27 mer DsiRNA, mismatch position=19 of sense strand (from5′-terminus)

(SEQ ID NO: 3460) 5′-AUAUUUUCCCAUCUGUA

AUUUtt-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAU

UAAAAA-5′Optionally, the mismatched ‘A’ residue of position 19 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=20 of sense strand (from5′-terminus)

(SEQ ID NO: 3461) 5′-AUAUUUUCCCAUCUGUAU

UUUtt-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUA

AAAAA-5′Optionally, the mismatched ‘U’ residue of position 20 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=21 of sense strand (from5′-terminus)

(SEQ ID NO: 3462) 5′-AUAUUUUCCCAUCUGUAUU

UUtt-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUAA

AAAA-5′Optionally, the mismatched ‘A’ residue of position 21 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=22 of sense strand (from5′-terminus)

(SEQ ID NO: 3463) 5′-AUAUUUUCCCAUCUGUAUUA

Utt-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUAAU

AAA-5′Optionally, the mismatched ‘G’ residue of position 22 of the sensestrand is alternatively ‘A’ or ‘C’.HAO1-1171 25/27 mer DsiRNA, mismatch position=23 of sense strand (from5′-terminus)

(SEQ ID NO: 3464) 5′-AUAUUUUCCCAUCUGUAUUAU

tt-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUAAUA

AA-5′Optionally, the mismatched ‘A’ residue of position 23 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=24 of sense strand (from5′-terminus)

(SEQ ID NO: 3465) 5′-AUAUUUUCCCAUCUGUAUUAUU

t-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUAAUAA

A-5′Optionally, the mismatched ‘g’ residue of position 24 of the sensestrand is alternatively ‘a’ or ‘c’.HAO1-1171 25/27 mer DsiRNA, mismatch position=25 of sense strand (from5′-terminus)

(SEQ ID NO: 3466) 5′-AUAUUUUCCCAUCUGUAUUAUUU

-3′ (SEQ ID NO: 580) 3′-GUUAUAAAAGGGUAGACAUAAUAAA

-5′Optionally, the mismatched ‘a’ residue of position 25 of the sensestrand is alternatively ‘c’ or ‘g’.HAO1-1171 25/27 mer DsiRNA, mismatch position=1 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAUUAUUU

-3′ (SEQ ID NO: 3467) 3′-GUUAUAAAAGGGUAGACAUAAUAAA

-5′Optionally, the mismatched ‘U’ residue of position 1 of the antisensestrand is alternatively ‘G’ or ‘C’.HAO1-1171 25/27 mer DsiRNA, mismatch position=2 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAUUAUU

-3′ (SEQ ID NO: 3468) 3′-GUUAUAAAAGGGUAGACAUAAUAA

A-5′Optionally, the mismatched ‘C’ residue of position 2 of the antisensestrand is alternatively ‘U’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=3 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAUUAU

tt-3′ (SEQ ID NO: 3469) 3′-GUUAUAAAAGGGUAGACAUAAUA

AA-5′Optionally, the mismatched ‘U’ residue of position 3 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=4 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAUUA

Utt-3′ (SEQ ID NO: 3470) 3′-GUUAUAAAAGGGUAGACAUAAU

AAA-5′Optionally, the mismatched ‘C’ residue of position 4 of the antisensestrand is alternatively ‘U’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=5 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAUU

UUtt-3′ (SEQ ID NO: 3471) 3′-GUUAUAAAAGGGUAGACAUAA

AAAA-5′Optionally, the mismatched ‘U’ residue of position 5 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=6 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUAU

UUUtt-3′ (SEQ ID NO: 3472) 3′-GUUAUAAAAGGGUAGACAUA

AAAAA-5′Optionally, the mismatched ‘A’ residue of position 6 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1171 25/27 mer DsiRNA, mismatch position=7 of antisense strand(from 5′-terminus)

(SEQ ID NO: 196) 5′-AUAUUUUCCCAUCUGUA

AUUUtt-3′ (SEQ ID NO: 3473) 3′-GUUAUAAAAGGGUAGACAU

UAAAAA-5′Optionally, the mismatched ‘U’ residue of position 7 of the antisensestrand is alternatively ‘C’ or ‘G’.

As another example, in the below structures, such mismatches areintroduced within the asymmetric HAO1-1378 DsiRNA (newly-introducedmismatch residues are italicized): HAO1-1378 25/27 mer DsiRNA, mismatchposition=19 of sense strand (from 5′-terminus)

(SEQ ID NO: 3474) 5′-UGGAAAUAUAUUAACUG

AAAAag-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGAC

UUUUUC-5′Optionally, the mismatched ‘A’ residue of position 19 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=20 of sense strand (from5′-terminus)

(SEQ ID NO: 3475) 5′-UGGAAAUAUAUUAACUGU

AAAag-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACA

UUUUC-5′Optionally, the mismatched ‘U’ residue of position 20 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=21 of sense strand (from5′-terminus)

(SEQ ID NO: 3476) 5′-UGGAAAUAUAUUAACUGUU

AAag-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACAA

UUUC-5′Optionally, the mismatched ‘U’ residue of position 21 of the sensestrand is alternatively ‘G’ or ‘C’.HAO1-1378 25/27 mer DsiRNA, mismatch position=22 of sense strand (from5′-terminus)

(SEQ ID NO: 3477) 5′-UGGAAAUAUAUUAACUGUUA

Aag-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACAAU

UUC-5′Optionally, the mismatched ‘G’ residue of position 22 of the sensestrand is alternatively ‘U’ or ‘C’.HAO1-1378 25/27 mer DsiRNA, mismatch position=23 of sense strand (from5′-terminus)

(SEQ ID NO: 3478) 5′-UGGAAAUAUAUUAACUGUUAA

ag-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACAAUU

UC-5′Optionally, the mismatched ‘U’ residue of position 23 of the sensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=24 of sense strand (from5′-terminus)

(SEQ ID NO: 3479) 5′-UGGAAAUAUAUUAACUGUUAAA

g-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACAAUUU

C-5′Optionally, the mismatched ‘g’ residue of position 24 of the sensestrand is alternatively ‘t’ or ‘c’.HAO1-1378 25/27 mer DsiRNA, mismatch position=25 of sense strand (from5′-terminus)

(SEQ ID NO: 3480) 5′-UGGAAAUAUAUUAACUGUUAAAA

-3′ (SEQ ID NO: 680) 3′-GGACCUUUAUAUAAUUGACAAUUUU

-5′Optionally, the mismatched ‘a’ residue of position 25 of the sensestrand is alternatively ‘t’ or ‘c’.HAO1-1378 25/27 mer DsiRNA, mismatch position=1 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGUUAAAA

-3′ (SEQ ID NO: 3481) 3′-GGACCUUUAUAUAAUUGACAAUUUU

-5′Optionally, the mismatched ‘U’ residue of position 1 of the antisensestrand is alternatively ‘A’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=2 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGUUAAA

g-3′ (SEQ ID NO: 3482) 3′-GGACCUUUAUAUAAUUGACAAUUU

C-5′Optionally, the mismatched ‘C’ residue of position 2 of the antisensestrand is alternatively ‘A’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=3 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGUUAA

ag-3′ (SEQ ID NO: 3483) 3′-GGACCUUUAUAUAAUUGACAAUU

UC-5′Optionally, the mismatched ‘A’ residue of position 3 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=4 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGUUA

Aag-3′ (SEQ ID NO: 3484) 3′-GGACCUUUAUAUAAUUGACAAU

UUC-5′Optionally, the mismatched ‘C’ residue of position 4 of the antisensestrand is alternatively ‘A’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=5 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGUU

AAag-3′ (SEQ ID NO: 3485) 3′-GGACCUUUAUAUAAUUGACAA

UUUC-5′Optionally, the mismatched ‘A’ residue of position 5 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=6 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUGU

AAAag-3′ (SEQ ID NO: 3486) 3′-GGACCUUUAUAUAAUUGACA

UUUUC-5′Optionally, the mismatched ‘A’ residue of position 6 of the antisensestrand is alternatively ‘C’ or ‘G’.HAO1-1378 25/27 mer DsiRNA, mismatch position=7 of antisense strand(from 5′-terminus)

(SEQ ID NO: 296) 5′-UGGAAAUAUAUUAACUG

AAAAag-3′ (SEQ ID NO: 3487) 3′-GGACCUUUAUAUAAUUGAC

UUUUUC-5′Optionally, the mismatched ‘U’ residue of position 7 of the antisensestrand is alternatively ‘C’ or ‘G’.

For the above oligonucleotide strand sequences, it is contemplated thatthe sense strand sequence of one depicted duplex can be combined with anantisense strand of another depicted duplex, thereby forming a distinctduplex—in certain instances, such duplexes contain a mismatched residuewith respect to the Glycolate Oxidase target transcript sequence, whilesuch sense and antisense strand sequences do not present a mismatch atthis residue with respect to one another (e.g., duplexes comprising SEQID NOs: 3466 and 3467; SEQ ID NOs: 3465 and 3468; SEQ ID NOs: 3464 and3469, etc., are contemplated as exemplary of such duplexes).

As noted above, introduction of mismatches can be performed upon any ofthe DsiRNAs described herein.

The mismatches of such DsiRNA structures can be combined to produce aDsiRNA possessing, e.g., two, three or even four mismatches within the3′-terminal four to seven nucleotides of the sense strand/5′-terminalfour to seven nucleotides of the antisense strand.

Indeed, in view of the flexibility of sequences which can beincorporated into DsiRNAs at the 3′-terminal residues of the sensestrand/5′-terminal residues of the antisense strand, in certainembodiments, the sequence requirements of an asymmetric DsiRNA of theinstant invention can be represented as the following (minimalist)structure (shown for an exemplary HAO1-1171 DsiRNA sequence):

(SEQ ID NO: 3488) 5′-AUAUUUUCCCAUCUGUAUXXXXXX[X]_(n)-3′(SEQ ID NO: 3489) 3′-GUUAUAAAAGGGUAGACAUAXXXXXX[X]_(n)-5′where n=1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 50, or 1 to 80 or more.

HAO1-1171 mRNA Target:  (SEQ ID NO: 3490)5′-CAAUAUUUUCCCAUCUGUAUXXXXXXX-3′.

The Glycolate Oxidase target site may also be a site which is targetedby one or more of several oligonucleotides whose complementary targetsites overlap with a stated target site. For example, for an exemplaryHAO1-1315 DsiRNA, it is noted that certain DsiRNAs targeting overlappingand only slightly offset Glycolate Oxidase sequences could exhibitactivity levels similar to that of HAO1-1315 (e.g., HAO1-1314 toHAO1-1317 of Table 2 above). Thus, in certain embodiments, a designatedtarget sequence region might be effectively targeted by a series ofDsiRNAs possessing largely overlapping sequences. (E.g., if consideringDsiRNAs of the HAO1-1314 to HAO1-1317 target site(s), a moreencompassing Glycolate Oxidase transcript target sequence might berecited as, e.g., 5′-TTTCATTGCTTTTGACTTTTCAATGGGTGT-3′ (SEQ ID NO:3491), wherein any given DsiRNA (e.g., a DsiRNA selected from HAO1-1314to HAO1-1317) only targets a sub-sequence within such a sequence region,yet the entire sequence can be considered a viable target for such aseries of DsiRNAs).

Additionally and/or alternatively, mismatches within the 3′-terminalseven nucleotides of the sense strand/5′-terminal seven nucleotides ofthe antisense strand can be combined with mismatches positioned at othermismatch-tolerant positions, as described above.

In view of the present identification of the above-described Dicersubstrate agents (DsiRNAs) as inhibitors of Glycolate Oxidase levels viatargeting of specific Glycolate Oxidase sequences, it is also recognizedthat dsRNAs having structures similar to those described herein can alsobe synthesized which target other sequences within the Glycolate Oxidasesequence of NM_017545.2, or within variants thereof (e.g., targetsequences possessing 80% identity, 90% identity, 95% identity, 96%identity, 97% identity, 98% identity, 99% or more identity to a sequenceof NM_017545.2).

Anti-Glycolate Oxidase DsiRNA Design/Synthesis

It has been found empirically that longer dsRNA species of from 25 to 35nucleotides (DsiRNAs) and especially from 25 to 30 nucleotides giveunexpectedly effective results in terms of potency and duration ofaction, as compared to 19-23 mer siRNA agents. Without wishing to bebound by the underlying theory of the dsRNA processing mechanism, it isthought that the longer dsRNA species serve as a substrate for the Dicerenzyme in the cytoplasm of a cell. In addition to cleaving the dsRNA ofthe invention into shorter segments, Dicer is thought to facilitate theincorporation of a single-stranded cleavage product derived from thecleaved dsRNA into the RISC complex that is responsible for thedestruction of the cytoplasmic RNA (e.g., Glycolate Oxidase RNA) of orderived from the target gene, Glycolate Oxidase (or other geneassociated with a Glycolate Oxidase-associated disease or disorder).Prior studies (Rossi et al., U.S. Patent Application No. 2007/0265220)have shown that the cleavability of a dsRNA species (specifically, aDsiRNA agent) by Dicer corresponds with increased potency and durationof action of the dsRNA species.

Certain anti-Glycolate Oxidase DsiRNA agents were selected from apre-screened population. Design of DsiRNAs can optionally involve use ofpredictive scoring algorithms that perform in silico assessments of theprojected activity/efficacy of a number of possible DsiRNA agentsspanning a region of sequence. Information regarding the design of suchscoring algorithms can be found, e.g., in Gong et al. (BMCBioinformatics 2006, 7:516), though a more recent “v4.3” algorithmrepresents a theoretically improved algorithm relative to siRNA scoringalgorithms previously available in the art. (E.g., “v3” and “v4” scoringalgorithms are machine learning algorithms that are not reliant upon anybiases in human sequence. In addition, the “v3” and “v4” algorithmsderive from data sets that are many-fold larger than that from which anolder “v2” algorithm such as that described in Gong et al. derives.)

The first and second oligonucleotides of the DsiRNA agents of theinstant invention are not required to be completely complementary. Infact, in one embodiment, the 3′-terminus of the sense strand containsone or more mismatches. In one aspect, two mismatches are incorporatedat the 3′ terminus of the sense strand. In another embodiment, theDsiRNA of the invention is a double stranded RNA molecule containing twoRNA oligonucleotides each of which is 27 nucleotides in length and, whenannealed to each other, have blunt ends and a two nucleotide mismatch onthe 3′-terminus of the sense strand (the 5′-terminus of the antisensestrand). The use of mismatches or decreased thermodynamic stability(specifically at the 3′-sense/5′-antisense position) has been proposedto facilitate or favor entry of the antisense strand into RISC (Schwarzet al., 2003, Cell 115: 199-208; Khvorova et al., 2003, Cell 115:209-216), presumably by affecting some rate-limiting unwinding stepsthat occur with entry of the siRNA into RISC. Thus, terminal basecomposition has been included in design algorithms for selecting active21 mer siRNA duplexes (Ui-Tei et al., 2004, Nucleic Acids Res 32:936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330). With Dicercleavage of the dsRNA of this embodiment, the small end-terminalsequence which contains the mismatches will either be left unpaired withthe antisense strand (become part of a 3′-overhang) or be cleavedentirely off the final 21-mer siRNA. These “mismatches”, therefore, donot persist as mismatches in the final RNA component of RISC. Thefinding that base mismatches or destabilization of segments at the3′-end of the sense strand of Dicer substrate improved the potency ofsynthetic duplexes in RNAi, presumably by facilitating processing byDicer, was a surprising finding of past works describing the design anduse of 25-30 mer dsRNAs (also termed “DsiRNAs” herein; Rossi et al.,U.S. Patent Application Nos. 2005/0277610, 2005/0244858 and2007/0265220).

Modification of Anti-Glycolate Oxidase dsRNAs

One major factor that inhibits the effect of double stranded RNAs(“dsRNAs”) is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) bynucleases. A 3′-exonuclease is the primary nuclease activity present inserum and modification of the 3′-ends of antisense DNA oligonucleotidesis crucial to prevent degradation (Eder et al., 1991, Antisense Res Dev,1: 141-151). An RNase-T family nuclease has been identified called ERI-1which has 3′ to 5′ exonuclease activity that is involved in regulationand degradation of siRNAs (Kennedy et al., 2004, Nature 427: 645-649;Hong et al., 2005, Biochem J, 390: 675-679). This gene is also known asThex1 (NM_026067) in mice or THEX1 (NM_153332) in humans and is involvedin degradation of histone mRNA; it also mediates degradation of3′-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al.,2006, J Biol Chem, 281: 30447-30454). It is therefore reasonable toexpect that 3′-end-stabilization of dsRNAs, including the DsiRNAs of theinstant invention, will improve stability.

XRN1 (NM_019001) is a 5′ to 3′ exonuclease that resides in P-bodies andhas been implicated in degradation of mRNA targeted by miRNA (Rehwinkelet al., 2005, RNA 11: 1640-1647) and may also be responsible forcompleting degradation initiated by internal cleavage as directed by asiRNA. XRN2 (NM_012255) is a distinct 5′ to 3′ exonuclease that isinvolved in nuclear RNA processing.

RNase A is a major endonuclease activity in mammals that degrades RNAs.It is specific for ssRNA and cleaves at the 3′-end of pyrimidine bases.SiRNA degradation products consistent with RNase A cleavage can bedetected by mass spectrometry after incubation in serum (Turner et al.,2007, Mol Biosyst 3: 43-50). The 3′-overhangs enhance the susceptibilityof siRNAs to RNase degradation. Depletion of RNase A from serum reducesdegradation of siRNAs; this degradation does show some sequencepreference and is worse for sequences having poly A/U sequence on theends (Haupenthal et al., 2006 Biochem Pharmacol 71: 702-710). Thissuggests the possibility that lower stability regions of the duplex may“breathe” and offer transient single-stranded species available fordegradation by RNase A. RNase A inhibitors can be added to serum andimprove siRNA longevity and potency (Haupenthal et al., 2007, Int J.Cancer 121: 206-210).

In 21 mers, phosphorothioate or boranophosphate modifications directlystabilize the internucleoside phosphate linkage. Boranophosphatemodified RNAs are highly nuclease resistant, potent as silencing agents,and are relatively non-toxic. Boranophosphate modified RNAs cannot bemanufactured using standard chemical synthesis methods and instead aremade by in vitro transcription (IVT) (Hall et al., 2004, Nucleic AcidsRes 32: 5991-6000; Hall et al., 2006, Nucleic Acids Res 34: 2773-2781).Phosphorothioate (PS) modifications can be easily placed in the RNAduplex at any desired position and can be made using standard chemicalsynthesis methods. The PS modification shows dose-dependent toxicity, somost investigators have recommended limited incorporation in siRNAs,favoring the 3′-ends where protection from nucleases is most important(Harborth et al., 2003, Antisense Nucleic Acid Drug Dev 13: 83-105; Chiuand Rana, 2003, Mol Cell 10: 549-561; Braasch et al., 2003, Biochemistry42: 7967-7975; Amarzguioui et al., 2003, Nucleic Acids Research 31:589-595). More extensive PS modification can be compatible with potentRNAi activity; however, use of sugar modifications (such as 2′-O-methylRNA) may be superior (Choung et al., 2006, Biochem Biophys Res Commun342: 919-927).

A variety of substitutions can be placed at the 2′-position of theribose which generally increases duplex stability (T_(m)) and cangreatly improve nuclease resistance. 2′-O-methyl RNA is a naturallyoccurring modification found in mammalian ribosomal RNAs and transferRNAs. 2′-O-methyl modification in siRNAs is known, but the preciseposition of modified bases within the duplex is important to retainpotency and complete substitution of 2′-O-methyl RNA for RNA willinactivate the siRNA. For example, a pattern that employs alternating2′-O-methyl bases can have potency equivalent to unmodified RNA and isquite stable in serum (Choung et al., 2006, Biochem Biophys Res Commun342: 919-927; Czauderna et al., 2003, Nucleic Acids Research 31:2705-2716).

The 2′-fluoro (2′-F) modification is also compatible with dsRNA (e.g.,siRNA and DsiRNA) function; it is most commonly placed at pyrimidinesites (due to reagent cost and availability) and can be combined with2′-O-methyl modification at purine positions; 2′-F purines are availableand can also be used. Heavily modified duplexes of this kind can bepotent triggers of RNAi in vitro (Allerson et al., 2005, J Med Chem 48:901-904; Prakash et al., 2005, J Med Chem 48: 4247-4253; Kraynack andBaker, 2006, RNA 12: 163-176) and can improve performance and extendduration of action when used in vivo (Morrissey et al., 2005, Hepatology41: 1349-1356; Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007). Ahighly potent, nuclease stable, blunt 19 mer duplex containingalternative 2′-F and 2′-O-Me bases is taught by Allerson. In thisdesign, alternating 2′-O-Me residues are positioned in an identicalpattern to that employed by Czauderna, however the remaining RNAresidues are converted to 2′-F modified bases. A highly potent, nucleaseresistant siRNA employed by Morrissey employed a highly potent, nucleaseresistant siRNA in vivo. In addition to 2′-O-Me RNA and 2′-F RNA, thisduplex includes DNA, RNA, inverted abasic residues, and a 3′-terminal PSinternucleoside linkage. While extensive modification has certainbenefits, more limited modification of the duplex can also improve invivo performance and is both simpler and less costly to manufacture.Soutschek et al. (2004, Nature 432: 173-178) employed a duplex in vivoand was mostly RNA with two 2′-O-Me RNA bases and limited 3′-terminal PSinternucleoside linkages.

Locked nucleic acids (LNAs) are a different class of 2′-modificationthat can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patternsof LNA incorporation that retain potency are more restricted than2′-O-methyl or 2′-F bases, so limited modification is preferred (Braaschet al., 2003, Biochemistry 42: 7967-7975; Grunweller et al., 2003,Nucleic Acids Res 31: 3185-3193; Elmen et al., 2005, Nucleic Acids Res33: 439-447). Even with limited incorporation, the use of LNAmodifications can improve dsRNA performance in vivo and may also alteror improve off target effect profiles (Mook et al., 2007, Mol CancerTher 6: 833-843).

Synthetic nucleic acids introduced into cells or live animals can berecognized as “foreign” and trigger an immune response. Immunestimulation constitutes a major class of off-target effects which candramatically change experimental results and even lead to cell death.The innate immune system includes a collection of receptor moleculesthat specifically interact with DNA and RNA that mediate theseresponses, some of which are located in the cytoplasm and some of whichreside in endosomes (Marques and Williams, 2005, Nat Biotechnol 23:1399-1405; Schlee et al., 2006, Mol Ther 14: 463-470). Delivery ofsiRNAs by cationic lipids or liposomes exposes the siRNA to bothcytoplasmic and endosomal compartments, maximizing the risk fortriggering a type 1 interferon (IFN) response both in vitro and in vivo(Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007; Sioud andSorensen, 2003, Biochem Biophys Res Commun 312: 1220-1225; Sioud, 2005,J Mol Biol 348: 1079-1090; Ma et al., 2005, Biochem Biophys Res Commun330: 755-759). RNAs transcribed within the cell are less immunogenic(Robbins et al., 2006, Nat Biotechnol 24: 566-571) and synthetic RNAsthat are immunogenic when delivered using lipid-based methods can evadeimmune stimulation when introduced unto cells by mechanical means, evenin vivo (Heidel et al., 2004, Nat Biotechnol 22: 1579-1582). However,lipid based delivery methods are convenient, effective, and widely used.Some general strategy to prevent immune responses is needed, especiallyfor in vivo application where all cell types are present and the risk ofgenerating an immune response is highest. Use of chemically modifiedRNAs may solve most or even all of these problems.

In certain embodiments, modifications can be included in theanti-Glycolate Oxidase dsRNA agents of the present invention so long asthe modification does not prevent the dsRNA agent from possessingGlycolate Oxidase inhibitory activity. In one embodiment, one or moremodifications are made that enhance Dicer processing of the DsiRNA agent(an assay for determining Dicer processing of a DsiRNA is describedelsewhere herein). In a second embodiment, one or more modifications aremade that result in more effective Glycolate Oxidase inhibition (asdescribed herein, Glycolate Oxidase inhibition/Glycolate Oxidaseinhibitory activity of a dsRNA can be assayed via art-recognized methodsfor determining RNA levels, or for determining Glycolate Oxidasepolypeptide levels, should such levels be assessed in lieu of or inaddition to assessment of, e.g., Glycolate Oxidase mRNA levels). In athird embodiment, one or more modifications are made that supportgreater Glycolate Oxidase inhibitory activity (means of determiningGlycolate Oxidase inhibitory activity are described supra). In a fourthembodiment, one or more modifications are made that result in greaterpotency of Glycolate Oxidase inhibitory activity per each dsRNA agentmolecule to be delivered to the cell (potency of Glycolate Oxidaseinhibitory activity is described supra). Modifications can beincorporated in the 3′-terminal region, the 5′-terminal region, in boththe 3′-terminal and 5′-terminal region or in some instances in variouspositions within the sequence. With the restrictions noted above inmind, numbers and combinations of modifications can be incorporated intothe dsRNA agent. Where multiple modifications are present, they may bethe same or different. Modifications to bases, sugar moieties, thephosphate backbone, and their combinations are contemplated. Either5′-terminus can be phosphorylated.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003,Nucleic Acids Research 31: 589-595). Examples of modificationscontemplated for the base groups include abasic sugars, 2-O-alkylmodified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's,could also be incorporated. Many other modifications are known and canbe used so long as the above criteria are satisfied. Examples ofmodifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988and 6,291,438 and in U.S. published patent application No. 2004/0203145A1. Other modifications are disclosed in Herdewijn (2000, AntisenseNucleic Acid Drug Dev 10: 297-310), Eckstein (2000, Antisense NucleicAcid Drug Dev 10: 117-21), Rusckowski et al. (2000, Antisense NucleicAcid Drug Dev 10: 333-345), Stein et al. (2001, Antisense Nucleic AcidDrug Dev 11: 317-25); Vorobjev et al. (2001, Antisense Nucleic Acid DrugDev 11: 77-85).

One or more modifications contemplated can be incorporated into eitherstrand. The placement of the modifications in the dsRNA agent cangreatly affect the characteristics of the dsRNA agent, includingconferring greater potency and stability, reducing toxicity, enhanceDicer processing, and minimizing an immune response. In one embodiment,the antisense strand or the sense strand or both strands have one ormore 2′-O-methyl modified nucleotides. In another embodiment, theantisense strand contains 2′-O-methyl modified nucleotides. In anotherembodiment, the antisense stand contains a 3′ overhang that is comprisedof 2′-O-methyl modified nucleotides. The antisense strand could alsoinclude additional 2′-O-methyl modified nucleotides.

In certain embodiments, the anti-Glycolate Oxidase DsiRNA agent of theinvention has several properties which enhance its processing by Dicer.According to such embodiments, the DsiRNA agent has a length sufficientsuch that it is processed by Dicer to produce an siRNA and at least oneof the following properties: (i) the DsiRNA agent is asymmetric, e.g.,has a 3′ overhang on the sense strand and (ii) the DsiRNA agent has amodified 3′ end on the antisense strand to direct orientation of Dicerbinding and processing of the dsRNA to an active siRNA. According tothese embodiments, the longest strand in the DsiRNA agent comprises25-30 nucleotides. In one embodiment, the sense strand comprises 25-30nucleotides and the antisense strand comprises 25-28 nucleotides. Thus,the resulting dsRNA has an overhang on the 3′ end of the sense strand.The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisensestrand may also have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing. Suitable modifiers include nucleotidessuch as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotidesand the like and sterically hindered molecules, such as fluorescentmolecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in dNMPs. Other nucleotide modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the sense strand.When sterically hindered molecules are utilized, they are attached tothe ribonucleotide at the 3′ end of the antisense strand. Thus, thelength of the strand does not change with the incorporation of themodifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the sense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of theantisense strand and the 3′ end of the sense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the antisensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

In certain other embodiments, the antisense strand of a DsiRNA agent ismodified for Dicer processing by suitable modifiers located at the 3′end of the antisense strand, i.e., the DsiRNA agent is designed todirect orientation of Dicer binding and processing. Suitable modifiersinclude nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of thesense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is also an asymmetric composition with DNA on the blunt endand RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the targetGlycolate Oxidase RNA.

Additionally, the DsiRNA agent structure can be optimized to ensure thatthe oligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention, a 27-bpoligonucleotide of the DsiRNA agent structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand.

US 2007/0265220 discloses that 27 mer DsiRNAs showed improved stabilityin serum over comparable 21 mer siRNA compositions, even absent chemicalmodification. Modifications of DsiRNA agents, such as inclusion of2′-O-methyl RNA in the antisense strand, in patterns such as detailedabove, when coupled with addition of a 5′ Phosphate, can improvestability of DsiRNA agents. Addition of 5′-phosphate to all strands insynthetic RNA duplexes may be an inexpensive and physiological method toconfer some limited degree of nuclease stability.

The chemical modification patterns of the dsRNA agents of the instantinvention are designed to enhance the efficacy of such agents.Accordingly, such modifications are designed to avoid reducing potencyof dsRNA agents; to avoid interfering with Dicer processing of DsiRNAagents; to improve stability in biological fluids (reduce nucleasesensitivity) of dsRNA agents; or to block or evade detection by theinnate immune system. Such modifications are also designed to avoidbeing toxic and to avoid increasing the cost or impact the ease ofmanufacturing the instant dsRNA agents of the invention.

In certain embodiments of the present invention, an anti-GlycolateOxidase DsiRNA agent has one or more of the following properties: (i)the DsiRNA agent is asymmetric, e.g., has a 3′ overhang on the antisensestrand and (ii) the DsiRNA agent has a modified 3′ end on the sensestrand to direct orientation of Dicer binding and processing of thedsRNA to an active siRNA. According to this embodiment, the longeststrand in the dsRNA comprises 25-35 nucleotides (e.g., 25, 26, 27, 28,29, 30, 31, 32, 33, 34 or 35 nucleotides). In certain such embodiments,the DsiRNA agent is asymmetric such that the sense strand comprises25-34 nucleotides and the 3′ end of the sense strand forms a blunt endwith the 5′ end of the antisense strand while the antisense strandcomprises 26-35 nucleotides and forms an overhang on the 3′ end of theantisense strand. In one embodiment, the DsiRNA agent is asymmetric suchthat the sense strand comprises 25-28 nucleotides and the antisensestrand comprises 25-30 nucleotides. Thus, the resulting dsRNA has anoverhang on the 3′ end of the antisense strand. The overhang is 1-4nucleotides, for example 2 nucleotides. The sense strand may also have a5′ phosphate.

The DsiRNA agent can also have one or more of the following additionalproperties: (a) the antisense strand has a right shift from the typical21 mer (e.g., the DsiRNA comprises a length of antisense strandnucleotides that extends to the 5′ of a projected Dicer cleavage sitewithin the DsiRNA, with such antisense strand nucleotides base pairedwith corresponding nucleotides of the sense strand extending 3′ of aprojected Dicer cleavage site in the sense strand), (b) the strands maynot be completely complementary, i.e., the strands may contain simplemismatched base pairs (in certain embodiments, the DsiRNAs of theinvention possess 1, 2, 3, 4 or even 5 or more mismatched base pairs,provided that Glycolate Oxidase inhibitory activity of the DsiRNApossessing mismatched base pairs is retained at sufficient levels (e.g.,retains at least 50% Glycolate Oxidase inhibitory activity or more, atleast 60% Glycolate Oxidase inhibitory activity or more, at least 70%Glycolate Oxidase inhibitory activity or more, at least 80% GlycolateOxidase inhibitory activity or more, at least 90% Glycolate Oxidaseinhibitory activity or more or at least 95% Glycolate Oxidase inhibitoryactivity or more as compared to a corresponding DsiRNA not possessingmismatched base pairs. In certain embodiments, mismatched base pairsexist between the antisense and sense strands of a DsiRNA. In someembodiments, mismatched base pairs exist (or are predicted to exist)between the antisense strand and the target RNA. In certain embodiments,the presence of a mismatched base pair(s) between an antisense strandresidue and a corresponding residue within the target RNA that islocated 3′ in the target RNA sequence of a projected Ago2 cleavage siteretains and may even enhance Glycolate Oxidase inhibitory activity of aDsiRNA of the invention) and (c) base modifications such as lockednucleic acid(s) may be included in the 5′ end of the sense strand. A“typical” 21 mer siRNA is designed using conventional techniques. In onetechnique, a variety of sites are commonly tested in parallel or poolscontaining several distinct siRNA duplexes specific to the same targetwith the hope that one of the reagents will be effective (Ji et al.,2003, FEBS Lett 552: 247-252). Other techniques use design rules andalgorithms to increase the likelihood of obtaining active RNAi effectormolecules (Schwarz et al., 2003, Cell 115: 199-208; Khvorova et al.,2003, Cell 115: 209-216; Ui-Tei et al., 2004, Nucleic Acids Res 32:936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330; Krol et al.,2004, J Biol Chem 279: 42230-42239; Yuan et al., 2004, Nucl Acids Res32(Webserver issue):W130-134; Boese et al., 2005, Methods Enzymol 392:73-96). High throughput selection of siRNA has also been developed (U.S.published patent application No. 2005/0042641 A1). Potential targetsites can also be analyzed by secondary structure predictions (Heale etal., 2005, Nucleic Acids Res 33(3): e30). This 21 mer is then used todesign a right shift to include 3-9 additional nucleotides on the 5′ endof the 21 mer. The sequence of these additional nucleotides is notrestricted. In one embodiment, the added ribonucleotides are based onthe sequence of the target gene. Even in this embodiment, fullcomplementarity between the target sequence and the antisense siRNA isnot required.

The first and second oligonucleotides of a DsiRNA agent of the instantinvention are not required to be completely complementary. They onlyneed to be sufficiently complementary to anneal under biologicalconditions and to provide a substrate for Dicer that produces a siRNAsufficiently complementary to the target sequence. Locked nucleic acids,or LNA's, are well known to a skilled artisan (Elmen et al., 2005,Nucleic Acids Res 33: 439-447; Kurreck et al., 2002, Nucleic Acids Res30: 1911-1918; Crinelli et al., 2002, Nucleic Acids Res 30: 2435-2443;Braasch and Corey, 2001, Chem Biol 8: 1-7; Bondensgaard et al., 2000,Chemistry 6: 2687-2695; Wahlestedt et al., 2000, Proc Natl Acad Sci USA97: 5633-5638). In one embodiment, an LNA is incorporated at the 5′terminus of the sense strand. In another embodiment, an LNA isincorporated at the 5′ terminus of the sense strand in duplexes designedto include a 3′ overhang on the antisense strand.

In certain embodiments, the DsiRNA agent of the instant invention has anasymmetric structure, with the sense strand having a 25-base pairlength, and the antisense strand having a 27-base pair length with a 2base 3′-overhang. In other embodiments, this DsiRNA agent having anasymmetric structure further contains 2 deoxynucleotides at the 3′ endof the sense strand in place of two of the ribonucleotides.

Certain DsiRNA agent compositions containing two separateoligonucleotides can be linked by a third structure. The third structurewill not block Dicer activity on the DsiRNA agent and will not interferewith the directed destruction of the RNA transcribed from the targetgene. In one embodiment, the third structure may be a chemical linkinggroup. Many suitable chemical linking groups are known in the art andcan be used. Alternatively, the third structure may be anoligonucleotide that links the two oligonucleotides of the DsiRNA agentin a manner such that a hairpin structure is produced upon annealing ofthe two oligonucleotides making up the dsRNA composition. The hairpinstructure will not block Dicer activity on the DsiRNA agent and will notinterfere with the directed destruction of the Glycolate Oxidase RNA.

Glycolate Oxidase cDNA and Polypeptide Sequences

Known human and mouse Glycolate Oxidase (HAO1) cDNA and polypeptidesequences include the following: human Hydroxyacid Oxidase 1 (HAO1)NM_017545.2 and corresponding human HAO1 polypeptide sequence GenBankAccession No. NP_060015.1; and mouse wild-type HAO1 sequence GenBankAccession No. NM_010403.2 (Mus musculus C57BL/6 HAO1) and correspondingmouse HAO1 sequence GenBank Accession No. NP_034533.1.

In Vitro Assay to Assess dsRNA Glycolate Oxidase Inhibitory Activity

An in vitro assay that recapitulates RNAi in a cell-free system can beused to evaluate dsRNA constructs targeting Glycolate Oxidase RNAsequence(s), and thus to assess Glycolate Oxidase-specific geneinhibitory activity (also referred to herein as Glycolate Oxidaseinhibitory activity) of a dsRNA. The assay comprises the systemdescribed by Tuschl et al., 1999, Genes and Development, 13, 3191-3197and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with dsRNA(e.g., DsiRNA) agents directed against Glycolate Oxidase RNA. ADrosophila extract derived from syncytial blastoderm is used toreconstitute RNAi activity in vitro. Target RNA is generated via invitro transcription from a selected Glycolate Oxidase expressing plasmidusing T7 RNA polymerase or via chemical synthesis. Sense and antisensedsRNA strands (for example, 20 uM each) are annealed by incubation inbuffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mMmagnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C.,then diluted in lysis buffer (for example 100 mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can bemonitored by gel electrophoresis on an agarose gel in TBE buffer andstained with ethidium bromide. The Drosophila lysate is prepared usingzero to two-hour-old embryos from Oregon R flies collected on yeastedmolasses agar that are dechorionated and lysed. The lysate iscentrifuged and the supernatant isolated. The assay comprises a reactionmixture containing 50% lysate [vol/vol], RNA (10-50 pM finalconcentration), and 10% [vol/vol] lysis buffer containing dsRNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which dsRNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [α-³²P] CTP, passed over a G50Sephadex column by spin chromatography and used as target RNA withoutfurther purification. Optionally, target RNA is 5′-³²P-end labeled usingT4 polynucleotide kinase enzyme. Assays are performed as described aboveand target RNA and the specific RNA cleavage products generated by RNAiare visualized on an autoradiograph of a gel. The percentage of cleavageis determined by PHOSPHOR IMAGER® (autoradiography) quantitation ofbands representing intact control RNA or RNA from control reactionswithout dsRNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in theGlycolate Oxidase RNA target for dsRNA mediated RNAi cleavage, wherein aplurality of dsRNA constructs are screened for RNAi mediated cleavage ofthe Glycolate Oxidase RNA target, for example, by analyzing the assayreaction by electrophoresis of labeled target RNA, or by northernblotting, as well as by other methodology well known in the art.

In certain embodiments, a dsRNA of the invention is deemed to possessGlycolate Oxidase inhibitory activity if, e.g., a 50% reduction inGlycolate Oxidase RNA levels is observed in a system, cell, tissue ororganism, relative to a suitable control. Additional metes and boundsfor determination of Glycolate Oxidase inhibitory activity of a dsRNA ofthe invention are described supra.

Conjugation and Delivery of Anti-Glycolate Oxidase dsRNA Agents

In certain embodiments, the present invention relates to a method fortreating a subject having or at risk of developing a disease or disorderfor which inhibition of Glycolate Oxidase is predicted to or has beendemonstrated to have therapeutic value (e.g., a liver disease such asprimary hyperoxaluria 1 (PH1)). In such embodiments, the dsRNA can actas novel therapeutic agents for controlling the disease or disorder forwhich inhibition of Glycolate Oxidase is predicted to or has beendemonstrated to have therapeutic value. The method comprisesadministering a pharmaceutical composition of the invention to thepatient (e.g., human), such that the expression, level and/or activityof a Glycolate Oxidase (HAO1) RNA is reduced. The expression, leveland/or activity of a polypeptide encoded by a Glycolate Oxidase (HAO1)RNA might also be reduced by a dsRNA of the instant invention, evenwhere said dsRNA is directed against a non-coding region of theGlycolate Oxidase transcript (e.g., a targeted 5′ UTR or 3′ UTRsequence). Because of their high specificity, the dsRNAs of the presentinvention can specifically target Glycolate Oxidase (HAO1) sequences ofcells and tissues, optionally even in an allele-specific manner wherepolymorphic alleles exist within an individual and/or population.

In the treatment of a disease or disorder for which inhibition ofGlycolate Oxidase is predicted to or has been demonstrated to havetherapeutic value, the dsRNA can be brought into contact with the cellsor tissue of a subject, e.g., the cells or tissue of a subjectexhibiting disregulation of Glycolate Oxidase and/or otherwise targetedfor reduction of Glycolate Oxidase levels. For example, dsRNAsubstantially identical to all or part of a Glycolate Oxidase RNAsequence may be brought into contact with or introduced into such acell, either in vivo or in vitro. Similarly, dsRNA substantiallyidentical to all or part of a Glycolate Oxidase RNA sequence may beadministered directly to a subject having or at risk of developing adisease or disorder for which inhibition of Glycolate Oxidase ispredicted to or has been demonstrated to have therapeutic value.

Therapeutic use of the dsRNA agents of the instant invention can involveuse of formulations of dsRNA agents comprising multiple different dsRNAagent sequences. For example, two or more, three or more, four or more,five or more, etc. of the presently described agents can be combined toproduce a formulation that, e.g., targets multiple different regions ofthe Glycolate Oxidase RNA, or that not only target Glycolate Oxidase RNAbut also target, e.g., cellular target genes associated with a diseaseor disorder for which inhibition of Glycolate Oxidase is predicted to orhas been demonstrated to have therapeutic value. A dsRNA agent of theinstant invention may also be constructed such that either strand of thedsRNA agent independently targets two or more regions of GlycolateOxidase RNA, or such that one of the strands of the dsRNA agent targetsa cellular target gene of Glycolate Oxidase known in the art.

Use of multifunctional dsRNA molecules that target more then one regionof a target nucleic acid molecule can also provide potent inhibition ofGlycolate Oxidase RNA levels and expression. For example, a singlemultifunctional dsRNA construct of the invention can target both theHAO1-1171 and HAO1-1378 sites simultaneously; additionally and/oralternatively, single or multifunctional agents of the invention can bedesigned to selectively target one splice variant of Glycolate Oxidaseover another.

Thus, the dsRNA agents of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treat,inhibit, reduce, or prevent a disease or disorder for which inhibitionof Glycolate Oxidase is predicted to or has been demonstrated to havetherapeutic value. For example, the dsRNA molecules can be administeredto a subject or can be administered to other appropriate cells evidentto those skilled in the art, individually or in combination with one ormore drugs under conditions suitable for the treatment.

The dsRNA molecules also can be used in combination with other knowntreatments to treat, inhibit, reduce, or prevent a disease or disorderfor which inhibition of Glycolate Oxidase is predicted to or has beendemonstrated to have therapeutic value in a subject or organism. Forexample, the described molecules could be used in combination with oneor more known compounds, treatments, or procedures to treat, inhibit,reduce, or prevent a disease or disorder for which inhibition ofGlycolate Oxidase is predicted to or has been demonstrated to havetherapeutic value in a subject or organism as are known in the art.

A dsRNA agent of the invention can be conjugated (e.g., at its 5′ or 3′terminus of its sense or antisense strand) or unconjugated to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye, cholesterol, or the like). Modifying dsRNA agentsin this way may improve cellular uptake or enhance cellular targetingactivities of the resulting dsRNA agent derivative as compared to thecorresponding unconjugated dsRNA agent, are useful for tracing the dsRNAagent derivative in the cell, or improve the stability of the dsRNAagent derivative compared to the corresponding unconjugated dsRNA agent.

In certain embodiments, specific exemplary forms of dsRNA conjugates arecontemplated. Notably, RNAi therapies, such as the dsRNAs that arespecifically exemplified herein, have demonstrated particularly goodability to be delivered to the cells of the liver in vivo (via, e.g.,lipid nanoparticles and/or conjugates such as dynamic polyconjugates orGalNAc conjugates—in certain exemplary embodiments, one or more GalNAcmoieties can be conjugated to a 3′- and/or 5′-overhang region of a dsNA,optionally to an “extended” overhang region of a dsNA (e.g., to a 5 ormore nucleotide, 8 or more nucleotide, etc. example of such anoverhang); additionally and/or alternatively, one or more GalNAcmoieties can be conjugated to ds extended regions of a dsNA, e.g., tothe duplex region formed by the 5′-end region of the guide/antisensestrand of a dsNA and the corresponding 3′-end region of thepassenger/sense strand of a dsNA and/or to the duplex region formed bythe 3′-end region of the guide/antisense strand of a dsNA and thecorresponding 5′-end region of the passenger/sense strand of a dsNA).Thus, formulated RNAi therapies, such as those described herein, areattractive modalities for treating or preventing diseases or disordersthat are present in, originate in or otherwise involve the liver.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

dsRNA agents of the invention may be directly introduced into a cell(i.e., intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the nucleic acid. Vascular or extravascular circulation, theblood or lymph system, and the cerebrospinal fluid are sites where thenucleic acid may be introduced.

The dsRNA agents of the invention can be introduced using nucleic aciddelivery methods known in art including injection of a solutioncontaining the nucleic acid, bombardment by particles covered by thenucleic acid, soaking the cell or organism in a solution of the nucleicacid, or electroporation of cell membranes in the presence of thenucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or otherwiseincrease inhibition of the target Glycolate Oxidase RNA.

A cell having a target Glycolate Oxidase RNA may be from the germ lineor somatic, totipotent or pluripotent, dividing or non-dividing,parenchyma or epithelium, immortalized or transformed, or the like. Thecell may be a stem cell or a differentiated cell. Cell types that aredifferentiated include adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands.

Depending on the particular target Glycolate Oxidase RNA sequence andthe dose of dsRNA agent material delivered, this process may providepartial or complete loss of function for the Glycolate Oxidase RNA. Areduction or loss of RNA levels or expression (either Glycolate OxidaseRNA expression or encoded polypeptide expression) in at least 50%, 60%,70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary.Inhibition of Glycolate Oxidase RNA levels or expression refers to theabsence (or observable decrease) in the level of Glycolate Oxidase RNAor Glycolate Oxidase RNA-encoded protein. Specificity refers to theability to inhibit the Glycolate Oxidase RNA without manifest effects onother genes of the cell. The consequences of inhibition can be confirmedby examination of the outward properties of the cell or organism or bybiochemical techniques such as RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS).Inhibition of target Glycolate Oxidase RNA sequence(s) by the dsRNAagents of the invention also can be measured based upon the effect ofadministration of such dsRNA agents upon development/progression of adisease or disorder for which inhibition of Glycolate Oxidase ispredicted to or has been demonstrated to have therapeutic value, e.g.,primary hyperoxaluria 1 (PH1) or other rare liver disease, either invivo or in vitro. Treatment and/or reductions in PH1 and/or other rareliver disease can include halting or reduction of organ damage (e.g.,kidney and/or liver damage) associated with PH1 or other rare liverdisease (e.g., reduction of kidney damage, liver damage, calcium oxalatedepositions, systemic oxalosis, osteolytic lesions, epiphysiolysis(e.g., in the bone of a subject), renal osteopathy, including renalcalcification and/or calcification/calcium oxalate depositions of theskin, eye or other organs of, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 99% or more, and can also be measured in logarithmicterms, e.g., 10-fold, 100-fold, 1000-fold, 10⁵-fold, 10⁶-fold, 10⁷-foldreduction in kidney damage, liver damage, calcium oxalate depositions,systemic oxalosis, osteolytic lesions, epiphysiolysis (e.g., in the boneof a subject), renal osteopathy, including renal calcification andcalcification/calcium oxalate depositions of the skin, eye or otherorgans could be achieved via administration of the dsRNA agents of theinvention to cells, a tissue, or a subject).

For RNA-mediated inhibition in a cell line or whole organism, expressionof a reporter or drug resistance gene whose protein product is easilyassayed can be measured. Such reporter genes include acetohydroxyacidsynthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ),beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracyclin. Depending on the assay, quantitation of theamount of gene expression allows one to determine a degree of inhibitionwhich is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to acell not treated according to the present invention.

Lower doses of injected material and longer times after administrationof RNA silencing agent may result in inhibition in a smaller fraction ofcells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targetedcells). Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target GlycolateOxidase RNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; RNA may be detected with a hybridization probehaving a nucleotide sequence outside the region used for the inhibitorydsRNA, or translated polypeptide may be detected with an antibody raisedagainst the polypeptide sequence of that region.

The dsRNA agent may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

Glycolate Oxidase Biology

Glycolate oxidase (the product of the HAO1 gene) is the enzymeresponsible for converting glycolate to glyoxylate in themitochondrial/peroxisomal glycine metabolism pathway. While glycolate isa harmless intermediate of the glycine metabolism pathway, accumulationof glyoxylate (via, e.g., AGT1 mutation) drives oxalate accumulation (ascalcium pricipitates in the kidney initially and other organseventually), which ultimately results in the PH1 disease.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for apharmaceutical composition comprising the dsRNA agent of the presentinvention. The dsRNA agent sample can be suitably formulated andintroduced into the environment of the cell by any means that allows fora sufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsRNA are known inthe art and can be used so long as the dsRNA gains entry to the targetcells so that it can act. See, e.g., U.S. published patent applicationNos. 2004/0203145 A1 and 2005/0054598 A1. For example, the dsRNA agentof the instant invention can be formulated in buffer solutions such asphosphate buffered saline solutions, liposomes, micellar structures, andcapsids. Formulations of dsRNA agent with cationic lipids can be used tofacilitate transfection of the dsRNA agent into cells. For example,cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(published PCT International Application WO 97/30731), can be used.Suitable lipids include Oligofectamine, Lipofectamine (LifeTechnologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.),or FuGene 6 (Roche) all of which can be used according to themanufacturer's instructions.

Such compositions typically include the nucleic acid molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; cHuh7ting agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in a selected solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The compounds can also be administered by transfection or infectionusing methods known in the art, including but not limited to the methodsdescribed in McCaffrey et al. (2002), Nature, 418(6893), 38-9(hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The compounds can also be administered by a method suitable foradministration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a nucleic acidmolecule (i.e., an effective dosage) depends on the nucleic acidselected. For instance, single dose amounts of a dsRNA (or, e.g., aconstruct(s) encoding for such dsRNA) in the range of approximately 1 pgto 1000 mg may be administered; in some embodiments, 10, 30, 100, or1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10,30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g ofthe compositions can be administered. The compositions can beadministered one from one or more times per day to one or more times perweek; including once every other day. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a nucleic acid (e.g., dsRNA), protein, polypeptide, or antibody caninclude a single treatment or, preferably, can include a series oftreatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The expression constructs may be constructs suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, e.g., Tuschl (2002, Nature Biotechnol 20:500-505).

It can be appreciated that the method of introducing dsRNA agents intothe environment of the cell will depend on the type of cell and the makeup of its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the dsRNA agents can be added directly to theliquid environment of the cells. Lipid formulations can also beadministered to animals such as by intravenous, intramuscular, orintraperitoneal injection, or orally or by inhalation or other methodsas are known in the art. When the formulation is suitable foradministration into animals such as mammals and more specificallyhumans, the formulation is also pharmaceutically acceptable.Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate dsRNA agents in a buffer or saline solution anddirectly inject the formulated dsRNA agents into cells, as in studieswith oocytes. The direct injection of dsRNA agent duplexes may also bedone. For suitable methods of introducing dsRNA (e.g., DsiRNA agents),see U.S. published patent application No. 2004/0203145 A1.

Suitable amounts of a dsRNA agent must be introduced and these amountscan be empirically determined using standard methods. Typically,effective concentrations of individual dsRNA agent species in theenvironment of a cell will be 50 nanomolar or less, 10 nanomolar orless, or compositions in which concentrations of 1 nanomolar or less canbe used. In another embodiment, methods utilizing a concentration of 200picomolar or less, 100 picomolar or less, 50 picomolar or less, 20picomolar or less, and even a concentration of 10 picomolar or less, 5picomolar or less, 2 picomolar or less or 1 picomolar or less can beused in many circumstances.

The method can be carried out by addition of the dsRNA agentcompositions to an extracellular matrix in which cells can live providedthat the dsRNA agent composition is formulated so that a sufficientamount of the dsRNA agent can enter the cell to exert its effect. Forexample, the method is amenable for use with cells present in a liquidsuch as a liquid culture or cell growth media, in tissue explants, or inwhole organisms, including animals, such as mammals and especiallyhumans.

The level or activity of a Glycolate Oxidase RNA can be determined by asuitable method now known in the art or that is later developed. It canbe appreciated that the method used to measure a target RNA and/or theexpression of a target RNA can depend upon the nature of the target RNA.For example, where the target Glycolate Oxidase RNA sequence encodes aprotein, the term “expression” can refer to a protein or the GlycolateOxidase RNA/transcript derived from the Glycolate Oxidase gene (eithergenomic or of exogenous origin). In such instances the expression of thetarget Glycolate Oxidase RNA can be determined by measuring the amountof Glycolate Oxidase RNA/transcript directly or by measuring the amountof Glycolate Oxidase protein. Protein can be measured in protein assayssuch as by staining or immunoblotting or, if the protein catalyzes areaction that can be measured, by measuring reaction rates. All suchmethods are known in the art and can be used. Where target GlycolateOxidase RNA levels are to be measured, art-recognized methods fordetecting RNA levels can be used (e.g., RT-PCR, Northern Blotting,etc.). In targeting Glycolate Oxidase RNAs with the dsRNA agents of theinstant invention, it is also anticipated that measurement of theefficacy of a dsRNA agent in reducing levels of Glycolate Oxidase RNA orprotein in a subject, tissue, in cells, either in vitro or in vivo, orin cell extracts can also be used to determine the extent of reductionof Glycolate Oxidase-associated phenotypes (e.g., disease or disorders,e.g., PH1, PH1-associated biomarkers and/or phenotypes, other rare liverdiseases and associated biomarkers and/or phenotypes, etc.). The abovemeasurements can be made on cells, cell extracts, tissues, tissueextracts or other suitable source material.

The determination of whether the expression of a Glycolate Oxidase RNAhas been reduced can be by a suitable method that can reliably detectchanges in RNA levels. Typically, the determination is made byintroducing into the environment of a cell undigested dsRNA such that atleast a portion of that dsRNA agent enters the cytoplasm, and thenmeasuring the level of the target RNA. The same measurement is made onidentical untreated cells and the results obtained from each measurementare compared.

The dsRNA agent can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a dsRNA agent andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a dsRNA agenteffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

Suitably formulated pharmaceutical compositions of this invention can beadministered by means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general, a suitable dosage unit of dsRNA will be in the range of0.001 to 0.25 milligrams per kilogram body weight of the recipient perday, or in the range of 0.01 to 20 micrograms per kilogram body weightper day, or in the range of 0.001 to 5 micrograms per kilogram of bodyweight per day, or in the range of 1 to 500 nanograms per kilogram ofbody weight per day, or in the range of 0.01 to 10 micrograms perkilogram body weight per day, or in the range of 0.10 to 5 microgramsper kilogram body weight per day, or in the range of 0.1 to 2.5micrograms per kilogram body weight per day. A pharmaceuticalcomposition comprising the dsRNA can be administered once daily.However, the therapeutic agent may also be dosed in dosage unitscontaining two, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day. In that case, the dsRNAcontained in each sub-dose must be correspondingly smaller in order toachieve the total daily dosage unit. The dosage unit can also becompounded for a single dose over several days, e.g., using aconventional sustained release formulation which provides sustained andconsistent release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.Regardless of the formulation, the pharmaceutical composition mustcontain dsRNA in a quantity sufficient to inhibit expression of thetarget gene in the animal or human being treated. The composition can becompounded in such a way that the sum of the multiple units of dsRNAtogether contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For acompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsRNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by Glycolate Oxidase (e.g.,normal functioning or misregulation and/or elevation of HAO1 transcriptand/or Glycolate Oxidase protein levels), or treatable via selectivetargeting of Glycolate Oxidase.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a dsRNA agent or vectoror transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above (including, e.g.,prevention of the commencement of, e.g., PH1-forming events within asubject via inhibition of Glycolate Oxidase expression), byadministering to the subject a therapeutic agent (e.g., a dsRNA agent orvector or transgene encoding same). Subjects at risk for the disease canbe identified by, for example, one or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the detection of, e.g., PH1 in a subject, orthe manifestation of symptoms characteristic of the disease or disorder,such that the disease or disorder is prevented or, alternatively,delayed in its progression.

Another aspect of the invention pertains to methods of treating subjectstherapeutically, i.e., altering the onset of symptoms of the disease ordisorder. These methods can be performed in vitro (e.g., by culturingthe cell with the dsRNA agent) or, alternatively, in vivo (e.g., byadministering the dsRNA agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target HAO1 RNAmolecules of the present invention or target HAO1 RNA modulatorsaccording to that individual's drug response genotype. Pharmacogenomicsallows a clinician or physician to target prophylactic or therapeutictreatments to patients who will most benefit from the treatment and toavoid treatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in a selected animal model. Forexample, a dsRNA agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, an agent (e.g., a therapeutic agent) can be used in ananimal model to determine the mechanism of action of such an agent.

Models Useful to Evaluate the Down-Regulation of HAO1 mRNA Levels andExpression

Cell Culture

The dsRNA agents of the invention can be tested for cleavage activity invivo, for example, using the following procedure. The nucleotidesequences within the HAO1 cDNA targeted by the dsRNA agents of theinvention are shown in the above HAO1 sequences.

The dsRNA reagents of the invention can be tested in cell culture usingHeLa or other mammalian cells (e.g., human cell lines Hep3B, HepG2,DU145, Calu3, SW480, T84, PL45, etc., and mouse cell lines Hepa1-6,AML12, Neuro2a, etc.) to determine the extent of HAO1 RNA and/orGlycolate Oxidase protein inhibition. In certain embodiments, DsiRNAreagents (e.g., see FIG. 1, and above-recited structures) are selectedagainst the HAO1 target as described herein. HAO1 RNA inhibition ismeasured after delivery of these reagents by a suitable transfectionagent to, for example, cultured HeLa cells or other transformed ornon-transformed mammalian cells in culture. Relative amounts of targetHAO1 RNA are measured by reporter assay (e.g., as exemplified below)and/or versus HPRT1, actin or other appropriate control using real-timePCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparisonis made to the activity of oligonucleotide sequences made to unrelatedtargets or to a randomized DsiRNA control with the same overall lengthand chemistry, or simply to appropriate vehicle-treated or untreatedcontrols. Primary and secondary lead reagents are chosen for the targetand optimization performed.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

For RT-qPCR assays, total RNA is prepared from cells following DsiRNAdelivery, for example, using Ambion Rnaqueous 4-PCR purification kit forlarge scale extractions, or Promega SV96 for 96-well assays. For Taqmananalysis, dual-labeled probes are synthesized with, for example, thereporter dyes FAM or VIC covalently linked at the 5′-end and thequencher dye TAMRA conjugated to the 3′-end. PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence detector using 50uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100mM reverse primer, 100 nM probe, 1×TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl2, 100 uM each dATP, dCTP, dGTP anddTTP, 0.2 U RNase Inhibitor (Promega), 0.025 U AmpliTaq Gold (PE-AppliedBiosystems) and 0.2 U M-MLV Reverse Transcriptase (Promega). The thermalcycling conditions can consist of 30 minutes at 48° C., 10 minutes at95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at60° C. Quantitation of target HAO1 mRNA level is determined relative tostandards generated from serially diluted total cellular RNA (300, 100,30, 10 ng/r×n) and normalizing to, for example, HPRT1 mRNA in eitherparallel or same tube TaqMan reactions.

Western Blotting

Cellular protein extracts can be prepared using a standard micropreparation technique (for example using RIPA buffer). Cellular proteinextracts are run on Tris-Glycine polyacrylamide gel and transferred ontomembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hours at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected on a VersaDoc imaging system

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, dsRNA molecules of the invention are complexed with cationiclipids for cell culture experiments. dsRNA and cationic lipid mixturesare prepared in serum-free OptimMEM (InVitrogen) immediately prior toaddition to the cells. OptiMEM is warmed to room temperature (about20-25° C.) and cationic lipid is added to the final desiredconcentration. dsRNA molecules are added to OptiMEM to the desiredconcentration and the solution is added to the diluted dsRNA andincubated for 15 minutes at room temperature. In dose responseexperiments, the RNA complex is serially diluted into OptiMEM prior toaddition of the cationic lipid.

Animal Models

The efficacy of anti-HAO1 dsRNA agents may be evaluated in an animalmodel. Animal models of PH1 as are known in the art can be used forevaluation of the efficacy, potency, toxicity, etc. of anti-HAO1 dsRNAs.Exemplary animal models useful for assessment of anti-HAO1 dsRNAsinclude mouse models of glycolate challenge and genetically engineeredmouse models of PH1 disease. Such animal models may also be used assource cells or tissue for assays of the compositions of the invention.Such models can additionally be used or adapted for use for pre-clinicalevaluation of the efficacy of dsRNA compositions of the invention inmodulating HAO1 gene expression toward therapeutic use.

Such models and/or wild-type mice can be used in evaluating the efficacyof dsRNA molecules of the invention to inhibit HAO1 levels, expression,development of HAO1-associated phenotypes, diseases or disorders, etc.These models, wild-type mice and/or other models can similarly be usedto evaluate the safety/toxicity and efficacy of dsRNA molecules of theinvention in a pre-clinical setting.

Specific examples of animal model systems useful for evaluation of theHAO1-targeting dsRNAs of the invention include wild-type mice andgenetically engineered alanine-glyoxylate aminotransferase-deficientmodel mice (see Salido et al. Proc. Natl. Acad. Sci. USA 103(48):18249-54). In an exemplary in vivo experiment, dsRNAs of the inventionare tail vein injected into such mouse models at doses ranging from 0.01to 0.1 to 1 mg/kg or, alternatively, repeated doses are administered atsingle-dose IC₅₀ levels, and organ samples (e.g., liver, but may alsoinclude prostate, kidney, lung, pancreas, colon, skin, spleen, bonemarrow, lymph nodes, mammary fat pad, etc.) are harvested 24 hours afteradministration of the final dose. Such organs are then evaluated formouse and/or human HAO1 levels, depending upon the model used, and/orfor impact upon PH1-related phenotypes. Duration of action can also beexamined at, e.g., 1, 4, 7, 14, 21 or more days after final dsRNAadministration.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1: Preparation of Double-Stranded RNA Oligonucleotides

Oligonucleotide Synthesis and Purification

DsiRNA molecules were designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. In presently exemplified agents, 384 human target HAO1sequences were selected for evaluation (a selection of the 384 humantarget HAO1 sites were predicted to be conserved with correspondingsites in the mouse HAO1 transcript sequence). The sequences of onestrand of the DsiRNA molecules were complementary to the target HAO1site sequences described above. The DsiRNA molecules were chemicallysynthesized using methods described herein. Generally, DsiRNA constructswere synthesized using solid phase oligonucleotide synthesis methods asdescribed for 19-23 mer siRNAs (see for example Usman et al., U.S. Pat.Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323;6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086;6,008,400; 6,111,086).

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies, Coralville, Iowa). Forexample, RNA oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques(Damha and Olgivie, 1993, Methods Mol Biol 20: 81-114; Wincott et al.,1995, Nucleic Acids Res 23: 2677-84). The oligomers were purified usingion-exchange high performance liquid chromatography (IE-HPLC) on anAmersham Source 15Q column (1.0 cm×25 cm; Amersham Pharmacia Biotech,Piscataway, N.J.) using a 15 min step-linear gradient. The gradientvaried from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A was100 mM Tris pH 8.5 and Buffer B was 100 mM Tris pH 8.5, 1 M NaCl.Samples were monitored at 260 nm and peaks corresponding to thefull-length oligonucleotide species were collected, pooled, desalted onNAP-5 columns, and lyophilized.

The purity of each oligomer was determined by capillary electrophoresis(CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.).The CE capillaries had a 100 μm inner diameter and contained ssDNA 100RGel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide wasinjected into a capillary, run in an electric field of 444 V/cm anddetected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urearunning buffer was purchased from Beckman-Coulter. Oligoribonucleotideswere obtained that were at least 90% pure as assessed by CE for use inexperiments described below. Compound identity was verified bymatrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectroscopy on a Voyager DE™ Biospectometry Work Station (AppliedBiosystems, Foster City, Calif.) following the manufacturer'srecommended protocol. Relative molecular masses of all oligomers wereobtained, often within 0.2% of expected molecular mass.

Preparation of Duplexes

Single-stranded RNA (ssRNA) oligomers were resuspended, e.g., at 100 μMconcentration in duplex buffer consisting of 100 mM potassium acetate,30 mM HEPES, pH 7.5. Complementary sense and antisense strands weremixed in equal molar amounts to yield a final solution of, e.g., 50 μMduplex. Samples were heated to 100° C. for 5′ in RNA buffer (IDT) andallowed to cool to room temperature before use. Double-stranded RNA(dsRNA) oligomers were stored at −20° C. Single-stranded RNA oligomerswere stored lyophilized or in nuclease-free water at −80° C.

Nomenclature

For consistency, the following nomenclature has been employed in theinstant specification. Names given to duplexes indicate the length ofthe oligomers and the presence or absence of overhangs. A “25/27” is anasymmetric duplex having a 25 base sense strand and a 27 base antisensestrand with a 2-base 3′-overhang. A “27/25” is an asymmetric duplexhaving a 27 base sense strand and a 25 base antisense strand.

Cell Culture and RNA Transfection

HeLa cells were obtained and maintained in DMEM (HyClone) supplementedwith 10% fetal bovine serum (HyClone) at 37° C. under 5% CO2. For RNAtransfections, cells were transfected with DsiRNAs at a finalconcentration of 1 nM or 0.1 nM using Lipofectamine™ RNAiMAX(Invitrogen) and following manufacturer's instructions. Briefly, for 0.1nM transfections, e.g., of Example 3 below, an aliquot of stock solutionof each DsiRNA was mixed with Opti-MEM I (Invitrogen) and Lipofectamine™RNAiMAX to reach a volume of 150 μL (with 0.3 nM DsiRNA). The resulting150 μL mix was incubated for 20 min at RT to allow DsiRNA:Lipofectamine™RNAiMAX complexes to form. Meanwhile, target cells were trypsinized andresuspended in medium. At the end of the 20 min of complexation, 50 uLof the DsiRNA:RNAiMAX mixture was added per well into triplicate wellsof 96 well plates. Finally, 100 μL of the cell suspension was added toeach well (final volume 150 μL) and plates were placed into theincubator for 24 hours.

Assessment of Glycolate Oxidase Inhibition

HAO1 target gene knockdown in human HeLa cells was determined byreporter assay (HeLa cells were transformed with a Psi-Check-HsHAO1plasmid and Renilla luciferase levels were then detected using aluminometer), while HAO1 target gene knockdown in mouse Hepa1-6 cellswas determined by qRT-PCR, with values normalized to HPRT and RPL23housekeeping genes, and to transfections with control DsiRNAs and/ormock transfection controls.

RNA Isolation and Analysis

Media was aspirated, and total RNA was extracted using the SV96 kit(Promega). Total RNA was reverse-transcribed using SuperscriptII, OligodT, and random hexamers following manufacturer's instructions.Typically, the resulting cDNA was analyzed by qPCR using primers andprobes specific for both the HAO1 gene and for the mouse genes HPRT-1and RPL23. An ABI 7700 was used for the amplification reactions. Eachsample was tested in triplicate. Relative Glycolate Oxidase RNA levelswere normalized to HPRT1 and RPL23 RNA levels and compared with RNAlevels obtained in transfection control samples.

Example 2: DsiRNA Inhibition of Glycolate Oxidase

DsiRNA molecules targeting Glycolate Oxidase were designed andsynthesized as described above and tested in human HeLa cells(alternatively, HepG2 or other human cells could have been used) forinhibitory efficacy. For transfection, annealed DsiRNAs were mixed withthe transfection reagent (Lipofectamine™ RNAiMAX, Invitrogen) andincubated for 20 minutes at room temperature. The HeLa (human) orHepa1-6 (mouse) cells (alternatively, mouse AML12 or other mouse cellscould have been used) were trypsinized, resuspended in media, and addedto wells (100 uL per well) to give a final DsiRNA concentration of 1 nMin a volume of 150 μl. Each DsiRNA transfection mixture was added to 3wells for triplicate DsiRNA treatments. Cells were incubated at 37° C.for 24 hours in the continued presence of the DsiRNA transfectionmixture. At 24 hours, human cells were subjected to luminometer assayfor detection of Renilla luciferase levels or RNA was prepared from eachwell of treated mouse cells. For such mouse cells, the supernatants withthe transfection mixtures were first removed and discarded, then thecells were lysed and RNA was prepared from each well. Target reporterluciferase levels (human) or HAO1 RNA levels (mouse) following treatmentwere evaluated by luminometer (human) or qRT-PCR (mouse) for the HAO1target gene, with values normalized to those obtained for controls.Triplicate data was averaged and the % error was determined for eachtreatment. Normalized data were both tablulated and graphed, and thereduction of target mRNA by active DsiRNAs in comparison to controls wasdetermined (see Table 11 below and FIGS. 2A to 2F).

TABLE 11 Glycolate Oxidase Inhibitory Efficacy of DsiRNAs Assayed at 1nM in Human HeLa and Mouse HEPA1-6 Cells Human-HeLa Human Psi-Check-HsHAO1 Plasmid Mouse-Hepa1-6 RLuc Remaining vs. Normalized HPRT/Rpl23;vs NC1, NC1, NC5, NC7 NC5, NC7 Hs HAO1 Reporter Mm HAO1 476- Mm HAO11775- Duplex Mm Macaque* Assay 611 (FAM) Assay 1888 (HEX) Assay NameLocation Location % Remaining % Remaining % Remaining HAO1-118 114 30.5± 7.1  88.3 ± 3.4  87.9 ± 4.2 HAO1-119 115 47.5 ± 2.7 102.7 ± 5.6  97.5± 7.2 HAO1-120 116 29.8 ± 5.8 100.5 ± 3.9  91.6 ± 7.6 HAO1-121 117 44.8± 3.6  96.4 ± 7.0 106.2 ± 6.6 HAO1-122 118 21.9 ± 0.9  83.2 ± 4.1  95.2± 10.2 HAO1-123 119 22.1 ± 6.4  95.5 ± 2.7 103.6 ± 8.7 HAO1-124 120 20.2± 1.2  82.2 ± 3.0  88.8 ± 5.9 HAO1-125 121 22.7 ± 2.4  86.6 ± 9.5  86.4± 4.0 HAO1-126 122 27.9 ± 11.0  77.7 ± 4.6  75.7 ± 5.0 HAO1-127 123 21.8± 5.0  97.9 ± 4.3  83.7 ± 6.2 HAO1-128 124 26.2 ± 3.8 101.7 ± 12.0  97.2± 14.4 HAO1-129 125 27.0 ± 2.0  92.8 ± 7.0  96.2 ± 13.6 HAO1-238 234 31033.7 ± 4.8  89.7 ± 6.4 119.2 ± 7.7 HAO1-239 235 311 57.3 ± 6.0  93.3 ±2.6 110.7 ± 9.3 HAO1-502 498 574 39.4 ± 5.8  72.5 ± 2.4  83.8 ± 1.9HAO1-503 499 575 40.0 ± 5.0  90.6 ± 7.3  82.3 ± 5.6 HAO1-583 579 65548.5 ± 12.6  42.5 ± 7.1  38.5 ± 7.1 HAO1-584 580 656 76.1 ± 2.3  45.5 ±5.7  38.3 ± 10.7 HAO1-753 749 825 37.8 ± 2.8  85.6 ± 9.6  90.7 ± 10.2HAO1-754 750 826 24.1 ± 1.4  40.8 ± 2.3  48.7 ± 6.4 HAO1-755 751 82725.8 ± 6.2  40.6 ± 5.8  51.8 ± 7.4 HAO1-756 752 828 38.6 ± 5.3  76.5 ±5.3  85.5 ± 9.4 HAO1-757 753 829 39.0 ± 3.6  70.0 ± 2.3  76.0 ± 3.4HAO1-758 754 830 32.6 ± 1.1  70.7 ± 8.3  65.5 ± 5.6 HAO1-759 755 83129.8 ± 2.7  40.0 ± 9.0  29.8 ± 13.2 HAO1-760 756 832 36.4 ± 3.2  62.6 ±10.7  60.7 ± 11.5 HAO1-761 757 833 42.6 ± 15.0 104.0 ± 8.5 111.7 ± 10.2HAO1-762 758 834 40.8 ± 2.0  58.2 ± 4.5  74.6 ± 3.7 HAO1-763 759 83548.6 ± 19.9  81.5 ± 5.9  91.8 ± 14.1 HAO1-806 802 878 22.9 ± 5.6  48.8 ±2.4  53.6 ± 4.9 HAO1-807 803 879 31.6 ± 7.3  42.4 ± 5.0  46.1 ± 2.4HAO1-808 804 880 53.2 ± 10.2  69.5 ± 6.0  61.3 ± 1.9 HAO1-809 805 88125.3 ± 6.4  24.8 ± 16.7  28.2 ± 19.1 HAO1-810 806 882 31.2 ± 4.7  40.2 ±5.4  45.0 ± 14.4 HAO1-811 807 883 45.1 ± 5.6  64.6 ± 6.4  73.2 ± 3.3HAO1-812 808 884 53.7 ± 2.4  74.6 ± 3.5  85.5 ± 8.6 HAO1-813 809 88551.0 ± 2.1  86.7 ± 5.7 120.4 ± 11.2 HAO1-814 810 886 36.0 ± 0.8  68.8 ±7.4  72.2 ± 17.9 HAO1-815 811 887 18.3 ± 5.9  22.8 ± 5.5  29.6 ± 4.9HAO1-886 882 51.6 ± 4.9  87.4 ± 11.4  88.1 ± 7.5 HAO1-887 883 33.3 ± 6.5 37.1 ± 3.9  34.0 ± 7.2 HAO1-1023 1019 1095 32.5 ± 7.9  41.4 ± 6.1  37.5± 2.5 HAO1-1024 1020 1096 35.8 ± 7.4  53.7 ± 17.7  42.6 ± 22.0 HAO1-10251021 1097 24.6 ± 5.4  27.8 ± 4.5  29.7 ± 9.7 HAO1-1026 1022 1098 32.6 ±4.6  48.1 ± 3.4  55.6 ± 11.8 HAO1-1027 1023 1099 21.7 ± 1.9  33.3 ± 3.0 36.3 ± 1.8 HAO1-1028 1024 1100 23.8 ± 2.5  32.1 ± 4.4  32.4 ± 8.6HAO1-1029 1025 18.1 ± 3.6  48.4 ± 9.8  48.8 ± 16.6 HAO1-1030 1026 26.0 ±4.6  30.4 ± 9.6  22.3 ± 26.6 HAO1-1031 1027 46.5 ± 1.1  47.8 ± 11.2 46.9 ± 15.0 HAO1-1032 1028 47.0 ± 3.3  32.4 ± 26.5  32.8 ± 23.0HAO1-1033 1029 44.0 ± 6.7  51.2 ± 23.8  55.6 ± 14.2 HAO1-1034 1030 31.0± 7.2  28.7 ± 27.0  33.2 ± 24.5 HAO1-1035 1031 31.5 ± 0.7  41.1 ± 5.0 45.6 ± 9.9 HAO1-1073 1069 22.7 ± 2.9  90.8 ± 4.7 104.2 ± 12.2 HAO1-10741070 17.0 ± 1.6 125.5 ± 3.4 100.0 ± 3.4 HAO1-1075 1071 20.6 ± 2.9  59.2± 5.6  52.3 ± 3.3 HAO1-1076 1072 36.1 ± 1.1  41.0 ± 4.2  40.5 ± 8.3HAO1-1077 1073 24.1 ± 3.3  88.5 ± 10.3  95.9 ± 7.7 HAO1-1078 1074 29.9 ±6.6  83.1 ± 6.4  89.4 ± 9.5 HAO1-1079 1075 17.3 ± 2.1  41.5 ± 8.4  51.3± 7.6 HAO1-1080 1076 19.8 ± 3.1  93.6 ± 8.7 105.2 ± 2.7 HAO1-1081 107720.1 ± 1.4  73.7 ± 4.5  88.5 ± 4.2 HAO1-1082 1078 19.4 ± 4.0 131.0 ±17.3 120.9 ± 7.0 HAO1-1083 1079 16.7 ± 5.3  42.8 ± 14.3  46.8 ± 16.5HAO1-1084 1080 22.2 ± 1.8  47.7 ± 4.3  45.0 ± 3.6 HAO1-1085 1081 115723.0 ± 6.7  52.4 ± 3.1  54.1 ± 2.6 HAO1-1086 1082 1158 17.1 ± 3.4  35.8± 4.1  39.9 ± 4.1 HAO1-1087 1083 1159 19.4 ± 3.3  25.3 ± 3.4  25.8 ± 7.7HAO1-1088 1084 1160 17.5 ± 2.1  46.7 ± 3.3  46.7 ± 4.4 HAO1-1089 10851161 21.2 ± 2.5  44.8 ± 6.5  45.1 ± 8.1 HAO1-1090 1086 1162 19.1 ± 2.8 67.8 ± 2.2  72.0 ± 4.0 HAO1-1091 1087 1163 21.1 ± 11.9  40.8 ± 5.5 30.2 ± 9.6 HAO1-1092 1088 1164 22.9 ± 2.5  43.2 ± 6.1  42.3 ± 7.6HAO1-1093 1089 1165 18.6 ± 3.5  31.2 ± 16.9  28.5 ± 15.2 HAO1-1094 10901166 20.8 ± 3.3  24.4 ± 10.3  26.8 ± 4.7 HAO1-1095 1091 1167 16.6 ± 4.1 37.6 ± 8.0  32.5 ± 18.5 HAO1-1096 1092 1168 25.1 ± 4.3  19.0 ± 5.5 19.0 ± 7.5 HAO1-1097 1093 1169 67.8 ± 4.1  19.8 ± 13.4  18.9 ± 2.5HAO1-1098 1094 1170 50.4 ± 2.6  33.3 ± 5.5  29.8 ± 1.8 HAO1-1099 10951171 32.7 ± 1.4  24.6 ± 5.4  22.3 ± 4.8 HAO1-1100 1096 1172 29.5 ± 1.0 30.3 ± 8.5  34.4 ± 9.2 HAO1-1101 1097 1173 51.9 ± 3.4  32.5 ± 6.2  35.1± 6.8 HAO1-1102 1098 1174 40.6 ± 1.4  84.1 ± 4.5  94.4 ± 8.3 HAO1-11031099 1175 38.9 ± 1.7  22.6 ± 9.9  30.5 ± 12.3 HAO1-1104 1100 1176 68.5 ±3.8  47.6 ± 8.6  47.8 ± 8.1 HAO1-1105 1101 1177 36.2 ± 3.8  20.1 ± 13.1 22.2 ± 25.5 HAO1-1106 1102 1178 49.5 ± 5.9  73.7 ± 6.3  74.8 ± 4.2HAO1-1107 1103 1179 35.9 ± 2.5  44.9 ± 6.0  38.6 ± 18.4 HAO1-1108 11041180 35.9 ± 3.0  69.0 ± 4.7  74.7 ± 10.4 HAO1-1109 1105 1181 30.2 ± 1.9 52.0 ± 8.4  50.7 ± 7.5 HAO1-1110 1106 1182 34.6 ± 4.6  25.1 ± 16.5 26.3 ± 7.1 HAO1-1111 1107 1183 26.0 ± 2.3  28.5 ± 4.5  32.9 ± 5.9HAO1-1112 1108 1184 34.2 ± 2.3  59.4 ± 8.8  65.3 ± 7.1 HAO1-1113 11091185 32.5 ± 2.1  29.3 ± 7.7  32.3 ± 12.1 HAO1-1114 1110 1186 39.4 ± 0.7 47.6 ± 11.3  50.9 ± 10.3 HAO1-1115 1111 1187 23.7 ± 1.6  45.0 ± 4.1 36.2 ± 5.3 HAO1-1116 1112 1188 28.0 ± 7.1  48.3 ± 8.1  34.2 ± 5.8HAO1-1117 1113 1189 29.9 ± 1.4  34.3 ± 14.9  29.9 ± 13.2 HAO1-1118 11141190 29.9 ± 2.8  29.1 ± 17.1  28.5 ± 15.6 HAO1-1119 1115 1191 36.2 ± 5.1 55.9 ± 9.1  48.9 ± 14.9 HAO1-1120 1116 1192 37.7 ± 4.0  54.1 ± 10.9 47.9 ± 6.8 HAO1-1121 1117 1193 26.8 ± 4.3  33.4 ± 4.6  31.9 ± 14.4HAO1-1122 1118 1194 27.4 ± 3.0  41.3 ± 4.3  32.1 ± 4.8 HAO1-1123 11191195 27.8 ± 5.4  36.4 ± 6.1  32.3 ± 3.2 HAO1-1124 1120 1196 30.5 ± 5.2 64.6 ± 8.8  56.3 ± 9.1 HAO1-1125 1121 1197 33.6 ± 4.2  37.6 ± 5.5  32.7± 12.1 HAO1-1126 1122 1198 26.4 ± 6.7  40.9 ± 3.0  41.2 ± 2.4 HAO1-11271123 1199 74.2 ± 5.1  78.6 ± 4.3  85.2 ± 5.7 HAO1-1147 1143 1219 38.3 ±4.1  56.8 ± 10.1  51.0 ± 13.8 HAO1-1148 1144 1220 36.8 ± 3.9  61.0 ± 7.1 58.6 ± 7.9 HAO1-1149 1145 1221 27.6 ± 1.5  33.6 ± 6.2  32.2 ± 0.3HAO1-1150 1146 1222 34.6 ± 1.1  60.2 ± 13.8  39.7 ± 8.2 HAO1-1151 11471223 36.7 ± 1.3  51.3 ± 5.6  44.6 ± 5.9 HAO1-1152 1148 1224 35.1 ± 2.5 42.7 ± 9.1  31.4 ± 10.7 HAO1-1153 1149 1225 32.7 ± 1.6  23.3 ± 4.2 23.2 ± 11.9 HAO1-1154 1150 1226 21.3 ± 8.3  23.2 ± 5.7  26.3 ± 5.1HAO1-1155 1151 1227 30.5 ± 0.9  42.0 ± 9.5  41.9 ± 7.8 HAO1-1156 11521228 27.8 ± 4.0  36.4 ± 6.3  38.6 ± 10.1 HAO1-1157 1153 1229 28.6 ± 0.9 36.8 ± 6.1  32.6 ± 5.6 HAO1-1158 1154 1230 30.1 ± 9.0  45.3 ± 3.8  38.5± 8.4 HAO1-1159 1155 1231 26.7 ± 1.5  43.8 ± 10.2  31.2 ± 4.9 HAO1-11601156 1232 45.8 ± 4.1  81.7 ± 7.9  62.2 ± 14.4 HAO1-1161 1157 1233 42.4 ±4.2  58.7 ± 9.5  56.6 ± 10.2 HAO1-1162 1158 1234 24.3 ± 1.9  27.4 ± 21.0 29.6 ± 16.6 HAO1-1163 1159 1235 22.7 ± 5.2  44.7 ± 8.9  43.1 ± 9.1HAO1-1164 1160 1236 27.6 ± 2.5  40.3 ± 9.9  35.4 ± 10.2 HAO1-1165 11611237 38.4 ± 2.1  61.6 ± 3.6  52.8 ± 7.7 HAO1-1166 1162 1238 29.9 ± 3.4 46.9 ± 5.5  44.3 ± 4.8 HAO1-1167 1163 1239 28.7 ± 3.6  41.3 ± 4.6  41.1± 5.7 HAO1-1168 1164 1240 27.9 ± 2.4  19.5 ± 10.7  12.2 ± 12.8 HAO1-11691165 1241 23.2 ± 4.0  21.1 ± 5.5  17.2 ± 7.4 HAO1-1170 1166 1242 27.3 ±5.0  23.9 ± 8.2  25.7 ± 17.1 HAO1-1171 1167 1243 23.4 ± 4.1  16.3 ± 9.6 15.8 ± 3.2 HAO1-1172 1168 1244 23.8 ± 0.6  23.1 ± 6.2  21.5 ± 7.8HAO1-1173 1169 1245 29.3 ± 0.8  35.7 ± 6.8  36.3 ± 9.4 HAO1-1207 12041280 48.6 ± 5.3  86.7 ± 21.6  52.9 ± 5.6 HAO1-1208 1205 1281 49.5 ± 2.7 47.3 ± 8.4  40.2 ± 5.8 HAO1-1209 1206 1282 48.9 ± 5.1  35.5 ± 4.9  29.8± 3.9 HAO1-1210 1207 1283 27.8 ± 1.6  17.3 ± 13.9  19.1 ± 9.6 HAO1-12111208 1284 32.3 ± 2.3  17.3 ± 5.5  21.5 ± 8.1 HAO1-1212 1209 1285 50.6 ±5.1  47.0 ± 1.6  48.4 ± 5.7 HAO1-1213 1210 1286 34.6 ± 3.1  35.9 ± 4.5 36.1 ± 1.9 HAO1-1214 1211 1287 62.1 ± 3.4  66.8 ± 6.9  62.1 ± 7.6HAO1-1215 1212 1288 31.0 ± 1.9  44.4 ± 24.2  34.7 ± 13.4 HAO1-1216 12131289 36.9 ± 4.3  41.3 ± 41.8  53.6 ± 42.5 HAO1-1217 1214 1290 35.0 ± 2.8 36.6 ± N/A  49.8 ± N/A HAO1-1218 1215 1291 41.1 ± 4.0  68.6 ± 48.3 49.0 ± 11.2 HAO1-1219 1216 1292 30.3 ± 0.6  20.3 ± 17.6  19.7 ± 26.5HAO1-1220 1217 1293 46.2 ± 2.9  74.7 ± 37.9  58.7 ± 20.3 HAO1-1221 12181294 25.6 ± 5.1  12.2 ± 26.8  16.4 ± 9.8 HAO1-1222 1219 1295 33.8 ± 8.7 25.7 ± 24.9  23.6 ± 1.8 HAO1-1223 1220 1296 47.9 ± 3.7  59.1 ± 22.4 48.4 ± 5.4 HAO1-1224 1221 1297 27.7 ± 3.5  32.8 ± 27.9  46.4 ± 25.0HAO1-1225 1222 1298 64.0 ± 3.0  51.3 ± N/A  63.2 ± N/A HAO1-1226 12231299 38.8 ± 6.2  14.0 ± 17.6  23.1 ± 5.6 HAO1-1227 1224 1300 44.1 ± 3.8 N/A ± N/A  N/A ± N/A HAO1-1228 1225 1301 60.2 ± 5.5  24.2 ± 14.9  44.7± 15.4 HAO1-1266 1263 20.7 ± 4.9  20.4 ± 30.4  30.2 ± 7.1 HAO1-1267 126426.5 ± 7.3  20.0 ± 4.6  21.9 ± 6.2 HAO1-1268 1265 27.5 ± 5.0  23.4 ±13.9  25.1 ± 11.0 HAO1-1269 1266 23.6 ± 2.8  86.3 ± 21.3  29.8 ± 7.7HAO1-1270 1267 30.9 ± 0.7 100.0 ± 48.0  32.0 ± 10.7 HAO1-1271 1268 24.9± 5.9  N/A ± N/A  N/A ± N/A HAO1-1272 1269 18.7 ± 1.3  8.0 ± N/A  12.3 ±N/A HAO1-1273 1270 23.1 ± 1.7  9.0 ± 11.8  10.8 ± 27.6 HAO1-1274 127125.4 ± 4.5  38.3 ± 54.0  25.4 ± 6.4 HAO1-1275 1272 25.6 ± 2.9  17.2 ±6.0  20.0 ± 6.3 HAO1-1276 1273 1349 26.4 ± 3.2  29.2 ± 13.8  22.3 ± 9.5HAO1-1277 1274 1350 24.7 ± 4.6  35.4 ± 3.6  30.3 ± 9.3 HAO1-1278 12751351 24.0 ± 1.3  24.7 ± 8.8  17.1 ± 17.3 HAO1-1279 1276 1352 22.1 ± 2.2 19.3 ± 10.6  19.8 ± 9.1 HAO1-1280 1277 1353 25.0 ± 2.5  35.5 ± 11.2 29.5 ± 5.9 HAO1-1281 1278 1354 27.3 ± 1.7  37.7 ± 11.3  33.0 ± 18.2HAO1-1282 1279 1355 23.6 ± 3.3  32.2 ± 6.4  33.4 ± 10.4 HAO1-1313 13101386 32.5 ± 7.9  47.6 ± 3.4  46.9 ± 3.5 HAO1-1314 1311 1387 18.2 ± 2.1 24.3 ± 15.9  17.9 ± 15.4 HAO1-1315 1312 1388 16.2 ± 0.6  24.9 ± 11.2 15.0 ± 11.1 HAO1-1316 1313 1389 17.1 ± 5.3  18.9 ± 4.2  12.6 ± 20.2HAO1-1317 1314 1390 20.9 ± 4.0  20.2 ± 4.5  20.7 ± 7.2 HAO1-1318 13151391 32.7 ± 1.6  44.2 ± 9.3  38.6 ± 3.0 HAO1-1319 1316 1392 33.7 ± 5.3 42.1 ± 14.2  42.5 ± 18.8 HAO1-1320 1317 1393 21.3 ± 3.0  24.0 ± 10.3 26.8 ± 8.4 HAO1-1321 1318 1394 29.6 ± 4.7  47.3 ± 6.3  57.1 ± 6.4HAO1-1322 1319 1395 22.0 ± 1.4  27.9 ± 19.7  30.7 ± 4.5 HAO1-1323 13201396 34.7 ± 3.2  61.3 ± 3.2  59.9 ± 3.6 HAO1-1324 1321 1397 40.9 ± 1.6 65.7 ± 11.9  57.4 ± 17.9 HAO1-1325 1322 1398 38.3 ± 2.2  45.5 ± 11.4 43.0 ± 26.3 HAO1-1326 1323 1399 24.4 ± 2.5  19.0 ± 7.0  19.8 ± 4.0HAO1-1327 1324 1400 25.9 ± 2.9  19.1 ± 7.0  16.3 ± 10.0 HAO1-1328 13251401 53.5 ± 6.4  46.6 ± 5.6  58.5 ± 5.7 HAO1-1329 1326 1402 26.0 ± 4.1 23.3 ± 6.5  26.6 ± 2.9 HAO1-1330 1327 1403 28.1 ± 10.3  27.5 ± 9.7 19.5 ± 11.5 HAO1-1331 1328 1404 26.5 ± 2.9  34.6 ± 12.9  24.4 ± 6.6HAO1-1332 1329 1405 25.5 ± 3.3  28.6 ± 24.6  22.8 ± 18.7 HAO1-1333 13301406 23.3 ± 1.7  22.4 ± 9.8  19.3 ± 17.6 HAO1-1334 1331 1407 29.6 ± 3.4 25.8 ± 18.2  24.2 ± 17.6 HAO1-1335 1332 1408 24.5 ± 0.4  21.2 ± 14.4 21.2 ± 14.0 HAO1-1373 1370 1446 30.2 ± 6.0  36.4 ± 7.4  31.7 ± 12.2HAO1-1374 1371 1447 25.4 ± 2.4  25.6 ± 5.3  17.5 ± 13.3 HAO1-1375 13721448 19.1 ± 4.9  20.7 ± 9.2  17.7 ± 14.4 HAO1-1376 1373 1449 22.5 ± 1.7 20.6 ± 4.3  18.5 ± 2.6 HAO1-1377 1374 1450 22.6 ± 7.0  23.8 ± 11.4 20.9 ± 19.7 HAO1-1378 1375 1451 19.8 ± 4.3  13.9 ± 4.2  12.4 ± 8.5HAO1-1379 1376 1452 18.5 ± 3.2  14.6 ± 5.7  14.7 ± 4.1 HAO1-1380 13771453 21.6 ± 5.8  22.9 ± 5.0  22.9 ± 3.7 HAO1-1381 1378 1454 24.9 ± 3.4 26.8 ± 7.4  29.6 ± 10.0 HAO1-1382 1379 1455 24.1 ± 4.6  26.1 ± 15.4 21.7 ± 9.3 HAO1-1383 1380 1456 26.1 ± 1.1  45.1 ± 27.8  23.9 ± 16.7HAO1-1384 1381 1457 25.2 ± 8.3  36.9 ± 14.1  32.1 ± 8.3 HAO1-1385 13821458 21.6 ± 2.7  23.7 ± 6.7  17.7 ± 9.1 HAO1-1386 1383 1459 28.2 ± 4.2 31.7 ± 2.4  30.1 ± 18.3 HAO1-1387 1384 1460 31.0 ± 1.4  37.6 ± 7.8 39.4 ± 1.8 HAO1-1388 1385 1461 33.6 ± 1.6  40.2 ± 4.1  49.7 ± 10.3HAO1-1389 1386 1462 33.8 ± 1.3  40.0 ± 7.5  41.1 ± 3.7 HAO1-1390 13871463 55.4 ± 6.1  69.1 ± 4.3  53.9 ± 10.0 HAO1-1391 1388 1464 28.1 ± 10.3 32.0 ± 6.1  21.5 ± 5.8 HAO1-1392 1389 1465 26.5 ± 2.9  28.7 ± 13.2 20.7 ± 25.5 HAO1-1393 1390 1466 25.5 ± 3.3  23.0 ± 21.0  13.3 ± 35.8HAO1-1394 1391 1467 23.3 ± 1.7  32.7 ± 17.1  35.5 ± 21.3 HAO1-1395 13921468 29.6 ± 3.4  42.7 ± 16.7  42.0 ± 5.0 HAO1-1426 1423 24.5 ± 0.4  79.5± 6.4  86.6 ± 5.4 HAO1-1427 1424 30.2 ± 6.0  88.6 ± 9.8  85.6 ± 21.4HAO1-1428 1425 25.4 ± 2.4  84.3 ± 4.2  58.9 ± 17.0 HAO1-1429 1426 19.1 ±4.9  69.0 ± 3.8  51.8 ± 6.6 HAO1-1430 1427 22.5 ± 1.7  92.6 ± 4.9  80.4± 8.6 HAO1-1431 1428 22.6 ± 7.0  49.2 ± 10.7  40.3 ± 23.4 HAO1-1432 142919.8 ± 4.3 108.3 ± 3.4 143.8 ± 5.0 HAO1-1433 1430 18.5 ± 3.2  47.7 ±17.1  58.3 ± 25.2 HAO1-1434 1431 21.6 ± 5.8  88.3 ± 0.7 111.8 ± 10.3HAO1-1435 1432 24.9 ± 3.4 102.7 ± 2.7 119.3 ± 3.2 HAO1-1436 1433 24.1 ±4.6 106.3 ± 11.1  85.7 ± 6.0 HAO1-1437 1434 26.1 ± 1.1  60.6 ± 14.5 74.4 ± 39.2 HAO1-1438 1435 25.2 ± 8.3  87.4 ± 5.4  88.5 ± 1.7 HAO1-59131 21.6 ± 2.7  92.2 ± 5.2 101.2 ± 12.5 HAO1-60 132 28.2 ± 4.2 105.6 ±16.4 110.7 ± 16.4 HAO1-61 133 31.0 ± 1.4 104.8 ± 3.9 112.6 ± 3.7 HAO1-62134 33.6 ± 1.6 113.9 ± 9.4 141.5 ± 11.6 HAO1-63 135 33.8 ± 1.3 116.1 ±3.9 136.4 ± 10.3 HAO1-64 136 55.4 ± 6.1 133.5 ± 6.3 107.4 ± 8.6 HAO1-65 8.1 ± 10.5 HAO1-66 12.2 ± 3.5 HAO1-67 11.0 ± 3.2 HAO1-68 17.4 ± 5.0HAO1-70 14.6 ± 5.5 HAO1-71 17.0 ± 1.0 HAO1-72 13.4 ± 2.0 HAO1-73 11.8 ±1.0 HAO1-74 19.6 ± 4.8 HAO1-75 16.3 ± 2.3 HAO1-76 20.5 ± 3.2 HAO1-7717.2 ± 3.0 HAO1-80 14.0 ± 1.6 HAO1-82 22.5 ± 1.3 HAO1-83 13.7 ± 0.9HAO1-86 15.0 ± 1.5 HAO1-95 18.1 ± 6.2 HAO1-96 18.0 ± 5.0 HAO1-98 21.3 ±8.4 HAO1-99 19.6 ± 5.3 HAO1-100 21.7 ± 1.2 HAO1-103 23.3 ± 0.5 HAO1-10622.9 ± 4.7 HAO1-107 18.4 ± 11.5 HAO1-109  8.1 ± 10.5 HAO1-212 12.2 ± 3.5HAO1-585 11.0 ± 3.2 HAO1-586 17.4 ± 5.0 HAO1-587 14.6 ± 5.5 HAO1-58917.0 ± 1.0 HAO1-590 13.4 ± 2.0 HAO1-591 11.8 ± 1.0 HAO1-592 19.6 ± 4.8HAO1-594 16.3 ± 2.3 HAO1-595 20.5 ± 3.2 HAO1-596 17.2 ± 3.0 HAO1-59714.0 ± 1.6 HAO1-598 22.5 ± 1.3 HAO1-599 13.7 ± 0.9 HAO1-600 15.0 ± 1.5HAO1-601 18.1 ± 6.2 HAO1-602 18.0 ± 5.0 HAO1-603 21.3 ± 8.4 HAO1-60419.6 ± 5.3 HAO1-605 21.7 ± 1.2 HAO1-606 23.3 ± 0.5 HAO1-607 22.9 ± 4.7HAO1-608 21.6 ± 15.8 HAO1-609 27.7 ± 2.4 HAO1-611 46.6 ± 1.5 HAO1-61247.5 ± 4.4 HAO1-621 41.0 ± 2.8 HAO1-716 30.0 ± 4.4 HAO1-857 31.3 ± 2.1HAO1-860 21.4 ± 2.0 HAO1-861 16.6 ± 2.8 HAO1-1194 22.0 ± 2.2 HAO1-119536.5 ± 0.8 HAO1-1196 25.3 ± 5.7 HAO1-1197 29.8 ± 5.3 HAO1-1198 17.7 ±0.4 HAO1-1199 19.7 ± 4.3 HAO1-1200 20.4 ± 2.3 HAO1-1201 19.6 ± 2.9HAO1-1202 17.0 ± 2.1 HAO1-1206 22.8 ± 1.5 HAO1-1246 22.6 ± 6.0 HAO1-124717.0 ± 2.3 HAO1-1248 18.8 ± 2.3 HAO1-1249 18.0 ± 2.4 HAO1-1289 21.1 ±2.7 HAO1-1290 19.1 ± 2.2 HAO1-1291 24.8 ± 5.1 HAO1-1292 19.4 ± 3.4HAO1-1293 21.8 ± 2.7 HAO1-1362 24.6 ± 1.8 HAO1-1363 27.1 ± 4.7 HAO1-136424.0 ± 1.0 HAO1-1365 29.7 ± 1.5 HAO1-1366 29.2 ± 4.4 HAO1-1367 30.4 ±3.9 HAO1-1368 28.4 ± 4.3 HAO1-1369 25.6 ± 2.9 HAO1-1370 26.5 ± 4.6HAO1-1371 18.5 ± 4.6 HAO1-1372 22.3 ± 3.3 HAO1-1478 24.8 ± 3.1 HAO1-147920.7 ± 3.5 HAO1-1480 18.5 ± 2.3 HAO1-1481 24.4 ± 3.3 HAO1-1483 25.0 ±3.7 HAO1-1484 24.4 ± 2.2 HAO1-1488 18.5 ± 2.7 HAO1-1489 16.1 ± 1.7HAO1-1490 14.1 ± 3.6 HAO1-1491 14.0 ± 8.6 HAO1-1492 17.5 ± 1.5 HAO1-149317.6 ± 2.9 HAO1-1494 15.9 ± 7.5 HAO1-1495 19.3 ± 2.4 HAO1-1496 15.6 ±4.8 HAO1-1497 16.5 ± 6.0 HAO1-1498 19.7 ± 2.2 HAO1-1499 15.6 ± 4.4HAO1-1500 15.3 ± 0.2 HAO1-1501 15.6 ± 2.6 HAO1-1502 18.4 ± 1.3 HAO1-150318.7 ± 2.3 HAO1-1504 14.6 ± 1.7 HAO1-1505 19.8 ± 2.1 HAO1-1506 34.9 ±3.4 HAO1-1507 16.2 ± 1.6 HAO1-1508 21.7 ± 2.5 HAO1-1509 30.5 ± 4.2HAO1-1510 14.7 ± 1.1 HAO1-1511 15.8 ± 0.8 HAO1-1512 14.7 ± 2.9 HAO1-151517.7 ± 3.4 HAO1-1516 26.8 ± 4.4 HAO1-1519 22.0 ± 1.5 HAO1-1520 23.3 ±4.2 HAO1-1546 17.0 ± 3.9 HAO1-1547 19.6 ± 1.8 HAO1-1548 16.9 ± 3.0HAO1-1549 16.3 ± 2.3 HAO1-1550 19.9 ± 4.6 HAO1-1551 24.3 ± 0.3 HAO1-155218.5 ± 0.8 HAO1-1557 16.4 ± 1.3 HAO1-1560 22.1 ± 3.7 HAO1-1561 16.8 ±3.4 HAO1-1563 16.8 ± 2.4 HAO1-1564 19.0 ± 2.1 HAO1-1565 18.8 ± 1.7HAO1-1656 18.7 ± 0.4 HAO1-1685 16.0 ± 2.9 HAO1-1695 19.9 ± 5.2 HAO1-169615.3 ± 2.1 HAO1-1697 17.9 ± 5.9 HAO1-1699 25.2 ± 1.6 HAO1-1700 19.8 ±1.8 HAO1-1701 19.9 ± 5.6 HAO1-1702 20.4 ± 1.0 HAO1-1703 17.1 ± 0.7*Macaca mulatta

Example 3: DsiRNA Inhibition of Glycolate Oxidase—Secondary Screen

96 asymmetric DsiRNAs (96 targeting Hs HAO1, 26 of which also targetedMm HAO1) of the above experiment were then examined in a secondary assay(“Phase 2”), with results of such assays presented in histogram form inFIGS. 3A to 3F. Specifically, the 96 asymmetric DsiRNAs selected fromthose tested above were assessed for inhibition of human HAO1 at 1 nM or0.1 nM (in duplicate assays) in the environment of human HeLa cells(FIGS. 3A and 3B). These 96 asymmetric DsiRNAs were also assessed forinhibition of mouse HAO1 at 1 nM or 0.1 nM (in duplicate) in theenvironment of mouse Hepa1-6 cells (FIGS. 3C to 3F). As shown in FIGS.3A and 3B, most asymmetric DsiRNAs reproducibly exhibited significanthuman HAO1 inhibitory efficacies at sub-nanomolar concentrations whenassayed in the environment of HeLa cells.

Without being bound by theory, it is noted that when using the RT-qPCRmethod to measure the amount of mRNA remaining after DsiRNA-mediatedmRNA knockdown, the position of the qPCR assay within the mRNA canaffect the detection of knockdown. The qPCR assay that is closer to theDsiRNA-directed mRNA cleavage point usually yields a more reliablemeasurement of mRNA level. For example, if a qPCR assay is located awayfrom the cleavage point, slow degradation of the mRNA fragment resultingfrom DsiRNA-directed cleavage can yield an artifactual high qPCR signal(a ‘false negative’ for knockdown). A sign of this is high knockdowndetected using a qPCR assay located near the DsiRNA site, andartifactual ‘low’ knockdown detected with a qPCR assay located away fromthe DsiRNA site. In this situation, the qPCR assay near the DsiRNA siteis used for quantitation.

As shown in FIGS. 3C to 3F, a number of asymmetric DsiRNAs were alsoidentified to possess significant mouse HAO1 inhibitory efficacies atsub-nanomolar concentrations when assayed in the environment of mouseHepa1-6 cells.

Example 4: Assessment of In Vivo Efficacy of Glycolate Oxidase-TargetingDsiRNAs

The ability of certain, active HAO1-targeting DsiRNAs to reduce HAO1levels within the liver of a mouse was examined. DsiRNAs employed in thestudy were: HAO1-1105, HAO1-1171, HAO1-1221, HAO1-1272, HAO1-1273,HAO1-1316, HAO1-1378 and HAO1-1379, each of which were synthesized withpassenger (sense) strand modification pattern “SM107” and guide(antisense) strand modification pattern “M48” (patterns describedabove). To perform the study, a primary hyperoxaluria model wasgenerated through oral gavage of 0.25 mL of 0.5 M glycolate to causeurine oxalate accumulation in C57BL/6 female mice. Animals wererandomized and assigned to groups based on body weight. Intravenousdosing of animals with lipid nanoparticles (LNPs; here, an LNPformulation named EnCore-2345 was employed) containing 1 mg/kg or 0.1mg/kg of DsiRNA was initiated on day 0. Dosing continued BIW for a totalof three doses in mice prior to glycolate challenge. Four hour and 24 hurine samples were collected after glycolate challenge for assessment ofoxalate/creatinine levels (see FIG. 4 for experimental flow chart).Animals were then sacrificed at 24 hrs after glycolate challenge. Liverwas dissected and weighed, and HAO1 levels were assessed using RT-qPCR,ViewRNA, western blot for glycolate oxidase and/or glycolate oxidaseimmunohistochemistry (ViewRNA, western blot for glycolate oxidase andglycolate oxidase immunohistochemistry data not shown). Serum sampleswere also subjected to ELISA for detection of glycolate oxidase (datanot shown). Notably, all eight DsiRNAs showed robust knockdown of HAO1when administered at 1 mg/kg (FIG. 5). At least two (HAO1-1171 andHAO1-1378) of the eight DsiRNAs tested in vivo also showed robustknockdown of HAO1 in all treated animals when administered at 0.1 mg/kg.As shown in FIG. 5, administration of the HAO1-1171-M107/M48 DsiRNA at0.1 mg/kg caused an average knockdown of 70% in liver tissue of treatedmice, while administration at 1 mg/kg produced an average knockdown of97% in liver tissue of treated mice. Similarly, administration of theHAO1-1378-M107/M48 DsiRNA at 0.1 mg/kg caused an average knockdown of53% in liver tissue of treated mice, while administration at 1 mg/kgproduced an average knockdown of 97% in liver tissue of treated mice.HAO-1171-induced knockdown at both 0.1 mg/kg and 1 mg/kg was furtherconfirmed by ViewRNA in situ hybridization assays.

Robust levels of HAO1 mRNA knockdown were observed in liver tissue ofmice treated with 1 mg/kg amounts of HAO1-targeting DsiRNAs HAO1-1171and HAO1-1378 (FIG. 6 and data not shown), and even 0.1 mg/kg amounts ofthese HAO1-targeting DsiRNAs produced robust HAO1 knockdown. As shown inFIG. 6, single dose HAO1-1171 DsiRNA treatment achieved durable HAO1mRNA target knockdown for at least 120 hours post-administration in theliver of treated animals. While robust HAO1 knockdown was achieved inliver, initial glycolate challenge experiments yielded inconclusivephenotypic results (data not shown).

In additional in vivo experiments, both HAO1 and oxalate levels wereassessed in both control- and DsiRNA-treated genetically engineered PH1model mice.

Example 5: Assessment of Modified Forms of Glycolate Oxidase-TargetingDsiRNAs In Vitro

A selection of 24 DsiRNAs from Example 3 above were prepared with2′-O-methyl guide strand modification patterns (“M17”, “M35”, “M48” and“M8” as shown above), paired with “M107” passenger strand modifications.For each of these DsiRNA sequences, DsiRNAs possessing each of the fourguide strand modification patterns M17, M35, M48 and M8 coupled with the“M107” passenger strand modification pattern were assayed for HAO1inhibition in human HeLa cells at 1.0 nM and 0.1 nM (in duplicate)concentrations in the environment of the HeLa cells. FIG. 7A showsspecific modification patterns for such duplexes (with shaded residuesindicating those possessing 2′-O-Methyl modifications), while FIGS. 7Band 7C show human HAO1 knockdown efficacy data for each of the fourmodified duplexes associated with the 24 duplex sequences examined(HAO1-65, 75, 77, 80, 83, 96, 1198, 1480, 1490, 1491, 1499, 1501, 1171,1279, 1504, 1549, 1552, 1696, 1315, 1316, 1375, 1378, 1379 and 1393). Asdemonstrated in FIGS. 7B and 7C, many tested duplexes were demonstratedto possess robust HAO1 knockdown efficacy at even 0.1 nM concentrationsacross the full range of highly modified forms tested.

Eight duplex sequences (HAO1-m1167, m1276, m1312, m1313, m1372, m1375,m1376 and m1390, which correspond to human duplexes HAO1-1171, 1279,1315, 1316, 1375, 1378, 1379 and 1393, respectively) were also examinedfor knockdown efficacy in mouse Hepa1-6 cells across the same range offour duplex modification patterns. As shown in FIGS. 7D and 7E, amajority of these duplexes showed robust HAO1 knockdown efficacy inmouse cells across a range of duplex modification patterns, atconcentrations as low as 0.03 nM.

Example 6: Assessment of Additionally Modified Forms of GlycolateOxidase-Targeting DsiRNAs In Vitro

The same set of 24 HAO1-targeting duplex sequences as set forth above inExample 5 were prepared to possess a range of four 2′-O-methyl passengerstrand modification patterns (“M107”, “M14”, “M24” and “M250”) coupledwith a fixed “M48” guide strand modification pattern. These DsiRNAs wereassayed for HAO1 inhibition at 1.0 nM and 0.1 nM (in duplicate)concentrations in the environment of the HeLa cells. FIG. 8A showsspecific modification patterns for such duplexes (with shaded residuesindicating those possessing 2′-O-Methyl modifications), while FIGS. 8Band 8C show human HAO1 knockdown efficacy data for each of the fourmodified duplexes associated with the 24 duplex sequences examined(HAO1-65, 75, 77, 80, 83, 96, 1198, 1480, 1490, 1491, 1499, 1501, 1171,1279, 1504, 1549, 1552, 1696, 1315, 1316, 1375, 1378, 1379 and 1393). Asdemonstrated in FIGS. 8B and 8C, many of the additional tested duplexeswere demonstrated to possess robust HAO1 knockdown efficacy at even 0.1nM concentrations across the full range of highly modified passengerstrand (with fixed guide strand modification pattern) forms tested.

Eight duplex sequences (HAO1-m1167, m1276, m1312, m1313, m1372, m1375,m1376 and m1390, which correspond to human duplexes HAO1-1171, 1279,1315, 1316, 1375, 1378, 1379 and 1393, respectively) were also examinedfor knockdown efficacy in mouse Hepa1-6 cells across the same range offour duplex modification patterns (with passenger strand modificationpatterns varied and guide strand modification pattern fixed, in contrastto the duplexes of Example 5 above). As shown in FIGS. 8D and 8E, amajority of these duplexes showed robust HAO1 knockdown efficacy inmouse cells across a range of duplex modification patterns, atconcentrations as low as 0.03 nM.

Example 7: Further Assessment of Modified Forms of GlycolateOxidase-Targeting DsiRNAs In Vitro

Further modification patterns of both passenger (sense) and guide(antisense) strands of the above 24 HAO1-targeting duplex sequences(HAO1-65, 75, 77, 80, 83, 96, 1171, 1198, 1279, 1315, 1316, 1375, 1378,1379, 1393, 1480, 1490, 1491, 1499, 1501, 1504, 1549, 1552 and 1696)were prepared to possess 2′-O-methyl modification patterns as shown inFIGS. 9A to 9D. These heavily modified DsiRNAs were assayed for HAO1inhibition at 1.0 nM and 0.1 nM (in duplicate) concentrations in theenvironment of either HeLa cells (using the luciferase reporter system,Psi-Check-HsHAO1 plasmid; FIGS. 9E to 9H) or human HEK293 cells stablytransfected with HAO1 (HEK293-pcDNA_HAO1 cells; FIGS. 91 to 9L). Asdemonstrated in FIGS. 9D to 9L, many of the additional tested duplexeswere identified to possess robust HAO1 knockdown efficacy at even 0.1 nMconcentrations across the full range of extensively modified formstested. Indeed, for all target sequences, one or more duplexespossessing extensive 2′-O-methyl modifications upon both strands wasidentified as a robust inhibitor of HAO1 expression. Additionally, theseresults confirmed the concordance between the luciferase system employedin many of the above examples (Psi-Check-HsHAO1 plasmid) and theHEK293-pcDNA_HAO1 cell stable transfectant system.

Example 8: Additional Extensively-Modified Forms of GlycolateOxidase-Targeting DsiRNAs were Active In Vitro

Four of the above 24 HAO1-targeting duplex sequences, HAO1-1171,HAO1-1315, HAO1-1378 and HAO1-1501, were prepared to possess extensivevariation in 2′-O-methyl modification patterns, as shown in FIGS. 10A to10K. These heavily modified DsiRNAs were assayed for HAO1 inhibition at1.0 nM and 0.1 nM (in duplicate) concentrations in the environment ofstably-transfected HEK293 cells (HEK293-pcDNA_HAO1 cells; FIGS. 10L to10O). As demonstrated in FIGS. 10L to 10O, the modified forms ofHAO1-1171, HAO1-1315, and HAO1-1501 duplexes were identified to possessrobust HAO1 knockdown efficacy at even 0.1 nM concentrations across thefull range of extensively modified forms tested. Meanwhile, for theHAO1-1378 duplex sequence, certain of the highly modified forms showedreduced inhibitory activities at 0.1 nM when compared with other heavilymodified forms (though a number of robustly active heavily 2′-O-methylmodified forms of HAO1-1378 duplexes were identified).

Modification of HAO1-1171, HAO1-1315, HAO1-1378 and HAO1-1501 duplexeswas expanded to include phosphorothioate linkages at the finalinternucleotide linkages of both the 5′ and 3′ ends of both guide andpassenger strands of variously 2′-O-methyl modified duplexes, as shownin FIGS. 11A to 11D. Five forms of each of the four duplexes weresynthesized on a small scale and were tested in a stable cell line(HAO1-6-9), at doses of 100 pM, 10 pM and 1 pM, with controls. As shownin FIG. 11E, significant knockdown was observed for all duplexes testedat 100 pM and even at 10 pM for the majority of duplexes. Of the 20forms examined, nine were selected for IC50 dose curve assessment, atconcentrations ranging from 10 nM to 10 fM, decreasing in ten-foldincrements, with results shown in FIG. 11F.

Modified forms of HAO1-1171 and HAO-1376 were further modified with2′-fluoro and inverted basic and additional and/or alternativelypositioned phosphorothioate (PS) modifications, layered upon a “parent”modification pattern, as shown in FIGS. 12A to 12D. As shown in FIGS.12E and 12F, highly active further modified (“derivative modificationpattern”) forms of each “parent” modification pattern presenting duplexwere identified, with significant efficacies observed at even 10 pMconcentrations.

Example 9: Glycolate Oxidase-Targeting DsiRNAs were Highly Active InVivo

Eight HAO1-targeting duplex sequences, HAO1-1105, HAO1-1171, HAO1-1221,HAO1-1272, HAO1-1273, HAO1-1316, HAO1-1378 and HAO1-1379, possessing anM107/M48 2′-O-methyl modification pattern, were formulated for in vivodelivery in a lipid nanoparticle (LNP) formulation (EnCore 2345). Asingle intravenous dose at 0.1 or 1.0 mg/kg was administered to mice(n=5 per group), and livers of treated animals were harvested at 24hours post-administration. The extent of HAO1 mRNA knockdown in treatedmouse liver was assessed by qPCR. As shown in FIG. 13A, all eightHAO1-targeting DsiRNAs tested for in vivo knockdown efficacy were activeat 1.0 mg/kg doses. Notably, HAO1-1171-M107/M48 and HAO1-1378-M107/M48duplexes exhibited robust knockdown of HAO1 in livers of treated miceeven at the 0.1 mg/kg dose (showing respective levels of knockdown ofapproximately 70% and approximately 53%).

To confirm the above qPCR results, and also to verify glycolate oxidaseprotein knockdown, ViewRNA assays and immunohistochemistry wereperformed upon liver samples of treated mice. As shown in FIG. 13B,HAO1-1171-treated mice showed significant HAO1 knockdown by both ViewRNA(top panels) and immunohistochemistry.

HAO1-targeting DsiRNAs were next examined for in vivo therapeuticefficacy in PH1 genetically engineered model mice. In AGXT^(−/−) PH1model mice, HAO1-targeting DsiRNA was initially intravenouslyadministered to mice at 0.3 mg/kg. At days 32 and 39, successiveadditional intravenous injections of HAO1-targeting DsiRNA wereadministered at 0.3 mg/kg, and the effect of this dosing regimen wasexamined for effect upon both the oxalate/creatinine ratio and theglycolate/creatinine ratio. As shown in FIG. 13C, each administration ofHAO1-targeting DsiRNA resulted in a marked decline in theoxalate/creatinine ratio of treated mice, while the glycolate/creatinineratio of the same mice was elevated following each DsiRNAadministration. Thus, formulated HAO1-targeting DsiRNA was effective atreducing the elevated oxalate levels that cause primary hyperoxaluriatype 1.

Having established the effect of HAO1-targeting DsiRNAs on theoxalate/creatinine ratio and the glycolate/creatinine ratio of treatedmice, it was then examined if HAO1-targeting DsiRNAs could also reduceurine glycolate oxidase levels in treated PH1 model mice. As shown inFIG. 13D, when formulated DsiRNAs were injected on days 1, 4, 6, 10 and14 for a total of 5 doses, into AGXT^(−/−) PH1 model mice, significantreductions of glycolate oxidase protein levels in liver harvested at day16 were observed.

Consistent with the above-observed effects of HAO1-targeting DsiRNAadministration, treatment of AGXT^(−/−) PH1 model mice withHAO1-targeting DsiRNA was observed to prevent kidney damage in such micewhen treated with ethylene glycol. As shown in FIG. 13E, miceadministered HAO1-targeting DsiRNA at three timepoints during ethyleneglycol administration were effectively protected from elevatedoxalate/creatinine ratios, which were observed in control miceadministered only PBS throughout ethylene glycol administration. Even asingle dose of a HAO1-targeting DsiRNA at the final administrationtimepoint succeeded in dramatically lowering the oxalate/creatinineratio to at or near wild type levels. Thus, kidney damage could beeffectively prevented or aggressively treated with an HAO1-targetingDsiRNA of the invention.

Histology confirmed the protective effect of HAO1-targeting DsiRNAs inthe AGXT^(−/−) PH1 mouse model. FIG. 13F shows the kidney damage thatwas elicited by feeding AGXT^(−/−) PH1 model mice with a 0.7% ethyleneglycol diet. As shown in FIGS. 13G and 13H, HAO1-targeting DsiRNAtreatment effectively maintained the kidneys of a treated animalthroughout ethylene glycol administration, while injection of HAO1 asonly the final dose of the regimen also caused a dramatic reduction inkidney damage (as compared to mice that received no HAO1 DsiRNAtreatment throughout ethylene glycol administration). Such effects wereobserved both in magnified sections (FIG. 13G) and at the level of thewhole kidney (FIG. 13H).

The kinetics and duration of in vivo HAO1 mRNA and protein (glycolateoxidase) inhibition were also examined. As shown in FIG. 13I, whenLNP-formulated HAO-targeting DsiRNA was injected at day 0 into C57BL/6mice at either 0.3 mg/kg or 1.0 mg/kg dosage, a dramatic reduction inlevels of HAO1 mRNA was immediately observed (FIG. 13I, top panel).Consistent with the glycolate oxidase protein having a short half-life(rapid turnover rate) in vivo, the dramatic inhibition of HAO1 mRNAobserved following administration of an HAO1-targeting DsiRNA produceddramatic reductions in glycolate oxidase protein levels over the 1-to-5day timeframe post-administration (FIG. 13I, lower panel, and FIG. 13J).Indeed, the half-life of the HAO1 protein in vivo was estimated to beless than 31 hours (based upon data from two sets of animals, assessedin FIG. 13J). The effect of HAO1-targeting DsiRNA on both HAO1 mRNA andglycolate oxidase protein levels in vivo was also highly durable—even attwenty-nine days after a single administration, mRNA levels weresignificantly reduced and no glycolate oxidase protein was detected bywestern analysis. Thus, HAO1 DsiRNA achieved a rapid and highly durablein vivo silencing effect on both HAO1 mRNA and protein production.

The silencing effect observed upon administration of a formulatedHAO1-targeting DsiRNA was also observed to be cumulative at low doselevels. As shown in FIG. 13K, HAO1 mRNA inhibition was documented to behighly responsive to HAO1-targeting DsiRNA in vivo, While responsivenessof treated mice to HAO1-targeting DsiRNA at the HAO1 protein level wasexpectedly dampened as compared to that observed at the mRNA level,responsiveness at the protein level was still observed at low doselevels, further confirming the robust in vivo impact of administeringLNP-formulated HAO1-targeting DsiRNAs to mice.

The in vivo efficacy of LNP-formulated HAO1-targeting DsiRNAs innon-human primates (NHPs) was also assessed. As shown in FIG. 13L,dramatic knockdown of both HAO1 mRNA and protein in the liver wasobserved in non-human primates administered an LNP-formulatedHAO1-targeting DsiRNA (HAO1-1171) at 0.3 mg/kg. Consistent with theabove-described mouse experiments, the inhibitory efficacy ofLNP-formulated HAO1-targeting DsiRNAs was observed to be rapid, robustand of extended duration in treated non-human primates. Indeed, at 29days post-administration, significant inhibitory efficacy persisted atboth the mRNA and protein levels in non-human primates.

Thus, HAO1 DsiRNA achieved a rapid and highly durable in vivo silencingeffect on both HAO1 mRNA and protein production in both mice andnon-human primates.

Example 10: Indications

The present body of knowledge in HAO1 research indicates the need formethods to assay HAO1 activity and for compounds that can regulate HAO1expression for research, diagnostic, and therapeutic use. As describedherein, the nucleic acid molecules of the present invention can be usedin assays to diagnose disease state related to HAO1 levels. In addition,the nucleic acid molecules can be used to treat disease state related toHAO1 functionality, misregulation, levels, etc.

Particular disorders and disease states that can be associated with HAO1expression modulation include, but are not limited to primaryhyperoxaluria 1 (PH1), including phenotypes of such disease in organssuch as kidney, eye, skin, liver, etc.

Other therapeutic agents can be combined with or used in conjunctionwith the nucleic acid molecules (e.g. DsiRNA molecules) of the instantinvention. Those skilled in the art will recognize that other compoundsand therapies used to treat the diseases and conditions described hereincan be combined with the nucleic acid molecules of the instant invention(e.g. siNA molecules, e.g., such as those directed to other enzymespossessing 2-hydroxyacid oxidase activity) and are hence within thescope of the instant invention. For example, for combination therapy,the nucleic acids of the invention can be prepared in one of at leasttwo ways. First, the agents are physically combined in a preparation ofnucleic acid and other agent, such as a mixture of a nucleic acid of theinvention encapsulated in liposomes and other agent in a solution forintravenous administration, wherein both agents are present in atherapeutically effective concentration (e.g., the other agent insolution to deliver 1000-1250 mg/m2/day and liposome-associated nucleicacid of the invention in the same solution to deliver 0.1-100mg/kg/day). Alternatively, the agents are administered separately butsimultaneously or successively in their respective effective doses(e.g., 1000-1250 mg/m2/d other agent and 0.1 to 100 mg/kg/day nucleicacid of the invention).

Example 11: Serum Stability for DsiRNAs

Serum stability of DsiRNA agents is assessed via incubation of DsiRNAagents in 50% fetal bovine serum for various periods of time (up to 24h) at 37° C. Serum is extracted and the nucleic acids are separated on a20% non-denaturing PAGE and can be visualized with Gelstar stain.Relative levels of protection from nuclease degradation are assessed forDsiRNAs (optionally with and without modifications).

Example 12: Diagnostic Uses

The DsiRNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of DsiRNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. DsiRNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells. The close relationship between DsiRNA activityand the structure of the target HAO1 RNA allows the detection ofmutations in a region of the HAO1 molecule, which alters thebase-pairing and three-dimensional structure of the target HAO1 RNA. Byusing multiple DsiRNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target HAO1 RNAswith DsiRNA molecules can be used to inhibit gene expression and definethe role of specified gene products in the progression of aHAO1-associated disease or disorder. In this manner, other genetictargets can be defined as important mediators of the disease. Theseexperiments will lead to better treatment of the disease progression byaffording the possibility of combination therapies (e.g., multipleDsiRNA molecules targeted to different genes, DsiRNA molecules coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of DsiRNA molecules and/or other chemical or biologicalmolecules). Other in vitro uses of DsiRNA molecules of this inventionare well known in the art, and include detection of the presence of RNAsassociated with a disease or related condition. Such RNA is detected bydetermining the presence of a cleavage product after treatment with aDsiRNA using standard methodologies, for example, fluorescence resonanceemission transfer (FRET).

In a specific example, DsiRNA molecules that cleave only wild-type ormutant or polymorphic forms of the target HAO1 RNA are used for theassay. The first DsiRNA molecules (i.e., those that cleave onlywild-type forms of target HAO1 RNA) are used to identify wild-type HAO1RNA present in the sample and the second DsiRNA molecules (i.e., thosethat cleave only mutant or polymorphic forms of target RNA) are used toidentify mutant or polymorphic HAO1 RNA in the sample. As reactioncontrols, synthetic substrates of both wild-type and mutant orpolymorphic HAO1 RNA are cleaved by both DsiRNA molecules to demonstratethe relative DsiRNA efficiencies in the reactions and the absence ofcleavage of the “non-targeted” HAO1 RNA species. The cleavage productsfrom the synthetic substrates also serve to generate size markers forthe analysis of wild-type and mutant HAO1 RNAs in the sample population.Thus, each analysis requires two DsiRNA molecules, two substrates andone unknown sample, which is combined into six reactions. The presenceof cleavage products is determined using an RNase protection assay sothat full-length and cleavage fragments of each HAO1 RNA can be analyzedin one lane of a polyacrylamide gel. It is not absolutely required toquantify the results to gain insight into the expression of mutant orpolymorphic HAO1 RNAs and putative risk of HAO1-associated phenotypicchanges in target cells. The expression of HAO1 mRNA whose proteinproduct is implicated in the development of the phenotype (i.e., diseaserelated/associated) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of HAO1 RNA levels is adequate and decreases thecost of the initial diagnosis. Higher mutant or polymorphic form towild-type ratios are correlated with higher risk whether HAO1 RNA levelsare compared qualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying DsiRNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170306333A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. (canceled)
 2. A nucleic acid comprising an oligonucleotide strand of19-80 nucleotides in length, wherein said oligonucleotide strand iscomplementary to SEQ ID NO: 1823 along at least 19 consecutivenucleotides of said oligonucleotide strand length and reduces HAO1target mRNA expression when said nucleic acid is introduced into amammalian cell.
 3. The nucleic acid of claim 2, wherein saidoligonucleotide strand is 19-35 nucleotides in length.
 4. (canceled) 5.A double stranded nucleic acid (dsNA) comprising first and secondnucleic acid strands, wherein said first strand is 15-66 nucleotides inlength and said second strand of said dsNA is 19-66 nucleotides inlength, wherein said second strand is complementary to SEQ ID NO: 1823along at least 19 consecutive nucleotides of said second oligonucleotidestrand length and reduces HAO1 target mRNA expression when said doublestranded nucleic acid is introduced into a mammalian cell. 6-7.(canceled)
 8. A double stranded nucleic acid (dsNA) comprising first andsecond nucleic acid strands and a duplex region of at least 25 basepairs, wherein said first strand is 25-65 nucleotides in length and saidsecond strand of said dsNA is 26-66 nucleotides in length and comprises1-5 single-stranded nucleotides at its 3′ terminus, wherein said secondstrand is complementary to SEQ ID NO: 1823 along at least 19 consecutivenucleotides of said second oligonucleotide strand length and reducesHAO1 target gene expression when said double stranded nucleic acid isintroduced into a mammalian cell.
 9. A double stranded nucleic acid(dsNA) comprising first and second nucleic acid strands and a duplexregion of at least 25 base pairs, wherein said first strand is 25-65nucleotides in length and said second strand of said dsNA is 26-66nucleotides in length and comprises 1-5 single-stranded nucleotides atits 3′ terminus, wherein the 3′ terminus of said first strand and the 5′terminus of said second strand form a blunt end, and said second strandis complementary to SEQ ID NO: 1823 along at least 19 nucleotides ofsaid second oligonucleotide strand length and reduces HAO1 mRNAexpression when said double stranded nucleic acid is introduced into amammalian cell.
 10. The dsNA of claim 5, wherein said first strand is15-35 nucleotides in length.
 11. The dsNA of claim 5, wherein saidsecond strand is 19-35 nucleotides in length.
 12. The dsNA of claim 5comprising a duplex region selected from the group consisting of atleast 25 base pairs; 19-21 base pairs and 21-25 base pairs.
 13. The dsNAof claim 5, wherein said second strand comprises 1-5 single-strandednucleotides at its 3′ terminus.
 14. The dsNA of claim 5, wherein saidsecond strand comprises 5-35 single-stranded nucleotides at its 3′terminus.
 15. The dsNA of claim 14, wherein said single-strandednucleotides comprise modified nucleotides.
 16. The dsNA of claim 14,wherein said single-stranded nucleotides comprise modified nucleotidesselected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy,2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio,4′-CH₂—O-2′-bridge, 4′-(CH₂)₂—O-2′-bridge, 2′-LNA, 2′-amino and2′-O—(N-methlycarbamate).
 17. The dsNA of claim 14, wherein saidsingle-stranded nucleotides comprise ribonucleotides.
 18. The dsNA ofclaim 14, wherein said single-stranded nucleotides comprisedeoxyribonucleotides.
 19. The dsNA of claim 5, wherein the 3′ end of thefirst strand and the 5′ end of the second strand are joined by apolynucleotide sequence comprising ribonucleotides, deoxyribonucleotidesor both, optionally wherein the polynucleotide sequence comprises atetraloop sequence.
 20. The dsNA of claim 5, wherein said first strandis 25-35 nucleotides in length.
 21. The dsNA of claim 5, wherein saidsecond strand is 25-35 nucleotides in length.
 22. (canceled)
 23. ThedsNA of claim 5, wherein starting from the first nucleotide (position 1)at the 3′ terminus of the first oligonucleotide strand, position 1, 2and/or 3 is substituted with a modified nucleotide.
 24. The dsNA ofclaim 5, wherein the 3′ terminus of said first strand and the 5′terminus of said second strand form a blunt end.
 25. The dsNA of claim5, wherein said first strand is 25 nucleotides in length and said secondstrand is 27 nucleotides in length.
 26. (canceled)
 27. The dsNA of claim5, wherein said second strand comprises a sequence comprising 23consecutive nucleotides of a sequence a set forth in SEQ ID NO: 5035.28. The dsNA of claim 5, wherein said first strand comprises a sequenceas set forth in SEQ ID NO:
 1823. 29-31. (canceled)
 32. The dsNA of claim14, wherein said nucleotides of said 5-35 single-stranded nucleotides ofsaid 3′ terminus of said second strand comprise a modified nucleotide.33. The dsNA of claim 32, wherein said modified nucleotide of said 5-35single-stranded nucleotides of said 3′ terminus of said second strand isa 2′-O-methyl ribonucleotide.
 34. The dsNA of claim 32, wherein allnucleotides of said 5-35 single-stranded nucleotides of said 3′ terminusof said second strand are modified nucleotides.
 35. The dsNA of claim 5,wherein said dsNA comprises a modified nucleotide.
 36. The dsNA of claim35, wherein said modified nucleotide residue is selected from the groupconsisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge,4′-(CH₂)₂—O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate).37. The dsNA of claim 13, wherein said 1-5 single-stranded nucleotidesof said 3′ terminus of said second strand are 1-3 nucleotides in length.38. The dsNA of claim 13, wherein said 1-5 single-stranded nucleotidesof said 3′ terminus of said second strand are 1-2 nucleotides in length.39. The dsNA of claim 13, wherein said 1-5 single-stranded nucleotidesof said 3′ terminus of said second strand is two nucleotides in lengthand comprises a 2′-O-methyl modified ribonucleotide. 40-42. (canceled)43. The dsNA of claim 5, wherein the amount of said nucleic acidsufficient to reduce expression of the target gene is selected from thegroup consisting of 1 nanomolar or less, 200 picomolar or less, 100picomolar or less, 50 picomolar or less, 20 picomolar or less, 10picomolar or less, 5 picomolar or less, 2, picomolar or less and 1picomolar or less in the environment of said cell.
 44. (canceled) 45.The nucleic acid of claim 2, wherein said nucleic acid reduces HAO1target mRNA expression by an amount (expressed by %) selected from thegroup consisting of at least 10%, at least 50%, at least 80-90%, atleast 95%, at least 98%, and at least 99% when said nucleic acid isintroduced into a mammalian cell.
 46. The dsNA of claim 5, wherein thefirst and second strands are joined by a chemical linker.
 47. The doublestranded nucleic acid of claim 5, wherein the 3′ terminus of said firststrand and the 5′ terminus of said second strand are joined by achemical linker.
 48. (canceled)
 49. The nucleic acid of claim 2comprising a modified nucleotide selected from the group consisting of adeoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a2′,3′-dideoxyinosine (ddl), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotideof 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine(3TC) and a monophosphate nucleotide of2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkylribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide,a 2′-fluoro ribonucleotide, and a locked nucleic acid.
 50. The nucleicacid of claim 2 comprising a phosphate backbone modification selectedfrom the group consisting of a phosphonate, a phosphorothioate and aphosphotriester.
 51. The nucleic acid of claim 2 comprising amodification selected from the group consisting of a morpholino nucleicacid and a peptide nucleic acid (PNA). 52-53. (canceled)
 54. The nucleicacid of claim 2, wherein said dsNA, nucleic acid or hybridizationcomplex is attached to a moiety selected from the group consisting of aGalNAc moiety, a cholesterol and a cholesterol targeting ligand.
 55. AdsNA comprising a first strand and a second strand, wherein the firststrand and the second strand form a duplex region of 19-25 nucleotidesin length, wherein the first strand comprises a 3′ region that extendsbeyond the first strand-second strand duplex region and comprises atetraloop, and the dsNA further comprises a discontinuity between the 3′terminus of the first strand and the 5′ terminus of the second strand,and the first or second strand is complementary to SEQ ID NO: 1823 alongat least 19 consecutive nucleotides of the first or second strand lengthand reduces HAO1 target mRNA expression when the dsNA is introduced intoa mammalian cell. 56-85. (canceled)